Multivalent macrolide antibiotics

ABSTRACT

Disclosed are multibinding compounds which include macrolide antibiotics, aminoglycosides, lincosamides, oxazolidinones, streptoramins, tetracycline and/or other compounds which bind to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium, which are useful in treating bacterial infections. The compounds adversely affect protein expression and have an antibacterial effect. The multibinding compounds of this invention containing from 2 to 10 ligands covalently attached to one or more linkers. Each ligand is macrolide antibiotic, aminoglycoside, lincosamide, oxazolidinone, streptogramin, tetracycline or other compound which binds to bacterial ribosomal RNA and/or one or more proteins involved in ribosomal protein synthesis in the bacterium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/327,899, filedJun. 8, 1999, now U.S. Pat. No. 6,566,509, which is hereby incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel multibinding compounds (agents) that aremacrolide antibiotics, aminoglycosides, lincosamides, oxazolidinones,streptogramins, tetracyclines or other compounds which bind to bacterialribosomal RNA or one or more proteins involved in ribosomal proteinsynthesis in the bacterium, and to pharmaceutical compositionscomprising such compounds. The compounds are useful as antibacterialagents for treating a variety of bacterial infections.

2. State of the Art

Organisms generate polypeptides (proteins) in order to survive.Organisms that cannot generate proteins cannot maintain viability.Because the majority of genes encode proteins, “gene expression” isnearly synonymous with protein synthesis. Gene expression involves twosteps—transcription and translation. Genes code for proteins usingvarious codons (units of three nucleotides), such as start codons (whichinitiate translation), stop codons (which stop translation) and codonsin between the start and stop codons which selectively code for thevarious amino acids.

Translation is the RNA directed synthesis of polypeptides. This processrequires all three classes of RNA. The template for correct addition ofindividual amino acids is the mRNA, yet both tRNAs and rRNAs areinvolved in the process.

Prokaryotes and eukaryotes use ribosomes to generate proteins. Ribosomesare cytoplasmic organelles, and are large complexes of proteins andthree (prokaryotes) or four (eukaryotes) rRNA (ribosomal ribonucleicacid) molecules called subunits made in the nucleolus. Ribosomes serveas the site of mRNA translation. Once the two (large and small) subunitsare joined by the mRNA from the nucleus, the ribosome translates themRNA into a specific sequence of amino acids, or a polypeptide chain.

In its inactive state, the ribosome exists as two subunits; a largesubunit and a small subunit. When the small subunit encounters an mRNA,the process of translation of the mRNA to protein begins. There are twosites in the large subunit, for subsequent amino acid-charged tRNAs tobind to and thus be close enough to each other for the formation of apeptide bond. The A site accepts a new tRNA bearing an amino acid, andthe P site bears the tRNA attached to the growing chain.

tRNA (transfer RNA) is a specific RNA molecule which acts as atranslator between mRNA and protein. Each tRNA has a specific anticodonand acceptor site. Each tRNA also has a specific charger protein; thisprotein can only bind to that particular tRNA and attach the correctamino acid to the acceptor site. The energy to make this bond comes fromATP. These charger proteins are called aminoacyl tRNA synthetases ThetRNAs carry activated amino acids into the ribosome. The ribosome isassociated with the mRNA ensuring correct access of activated tRNAs andcontaining the necessary enzymatic activities to catalyze peptide bondformation.

Protein synthesis proceeds from the N-terminus to the C-terminus of theprotein. The ribosomes “read” the mRNA in the 5′ to 3′ direction. Activetranslation occurs on polyribosomes (also termed polysomes). This meansthat more than one ribosome can be bound to and translate a given mRNAat any one time. Chain elongation occurs by sequential addition of aminoacids to the C-terminal end of the ribosome bound polypeptide.Translation proceeds in an ordered process. First accurate and efficientinitiation occurs, then chain elongation and finally accurate andefficient termination must occur. All three of these processes requirespecific proteins, some of which are ribosome associated and some ofwhich are separate from the ribosome, but may be temporarily associatedwith it.

Initiation of Translation

Before translation occurs, a ribosome must dissociate into its 30S and50S subunits. A ternary complex termed the preinitiation complex isformed consisting of the initiator, GTP, eIF-2 and the 40S subunit. ThemRNA is bound to the preinitiation complex.

Initiation of translation in both prokaryotes and eukaryotes requires aspecific initiator tRNA (which includes methionine). The initiation oftranslation requires recognition of a start (AUG) codon.

Peptide bond formation is catalyzed by a 23S rRNA component of the 50Ssubunit (peptidyl transferase). Another important site for proteinsynthesis is the 16S rRNA component of the 30S subunit (the aminoacyltRNA site). When protein synthesis is terminated, release factorproteins bind to the stop codons, GTP hydrolysis occurs, and peptidyltransferase activity is stimulated, causing release of the protein fromthe tRNA.

Elongation of the New Protein

After the first charged tRNA appears in the A site, the ribosome shiftsso that the tRNA is in the P site. New charged tRNAs, corresponding tothe codons of the mRNA, enter the A site, and a peptide bond is formedbetween the two amino acids. The first tRNA is now released and theribosome shifts again so that a tRNA carrying two amino acids is now inthe P site, and a new charged tRNA can bind to the A site. This processof elongation continues until the ribosome reaches a stop codon.

Elongation requires specific non-ribosomal proteins. Elongation ofpolypeptides occurs in a cyclic manner. At the end of one complete roundof amino acid addition the A site will be empty and ready to accept theincoming aminoacyl-tRNA dictated by the next codon of the mRNA. Thismeans that not only does the incoming amino acid need to be attached tothe peptide chain but the ribosome must move down the mRNA to the nextcodon. Each incoming aminoacyl-tRNA is brought to the ribosome by aneEF-1a-GTP complex. When the correct tRNA is deposited into the A sitethe GTP is hydrolyzed and the eEF-1a-GDP complex dissociates. Foradditional translocation events to occur the GDP must be exchanged forGTP. This is carried out by eEF-1bg similarly to the GTP exchange thatoccurs with eIF-2 catalyzed by eIF-2B. The peptide attached to the tRNAin the P site is transferred to the amino group at the aminoacyl-tRNA inthe A site. This reaction is catalyzed by peptidyltransferase in aprocess termed transpeptidation. The elongated peptide now resides on atRNA in the A site. The A site needs to be freed in order to accept thenext aminoacyl-tRNA. The process of moving the peptidyl-tRNA from the Asite to the P site is termed, translocation. Translocation is catalyzedby eEF-2 coupled to GTP hydrolysis. In translocation, the ribosome ismoved along the mRNA such that the next codon of the mRNA resides underthe A site. Following translocation, eEF-2 is released from the ribosomeand the cycle can start over again.

Termination of the Protein

When the ribosome reaches a stop codon, no aminoacyl tRNA binds to theempty A site. This signals the ribosomes to break into its large andsmall subunits, releasing the new protein and the mRNA. The protein maythen undergo post-translational modifications. For example, it might becleaved by a proteolytic (protein-cutting) enzyme at a specific place,have some of its amino acids altered, or become phosphorylated orglycosylated.

Protein Synthesis Inhibitors

In bacteria, if the ribosomal RNA is inactivated, for example, throughbinding of a ligand to the ribosomal RNA, protein synthesis is adverselyaffected and the bacteria will likely die. Many of the antibiotics, inparticular, MLS antibiotics, used to treat bacterial infections functionby inhibiting translation. Inhibition can be effected at all stages oftranslation, from initiation to elongation to termination. Manyantibiotics are believed to primarily attach to the ribosomal RNA at the16S and 23S ribosomal subunits and inhibit growth of bacteria byinhibiting protein synthesis.

Chloramphenicol inhibits prokaryotic peptidyl transferase. Streptomycinand neomycin inhibit prokaryotic peptide chain initiation, also inducemRNA misreading. Tetracycline inhibits prokaryotic aminoacyl-tRNAbinding to the ribosome small subunit. Erythromycin inhibits prokaryotictranslocation through the ribosome large subunit, and fusidic acidfunctions in a manner similar to erythromycin.

MLS antibiotics such as erythromycin have been used for years to treatvarious infections, for example, those caused by gram positive bacteria,gram negative bacteria and anaerobic bacteria. Many bacteria arebecoming drug resistant. One theory of how the bacteria become drugresistant is that their ribosomal RNA mutates such that the antibioticsno longer bind as ligands to the RNA.

A number of macrolide antibiotics are orally bioavailable, but incontact with the gastrointestinal tract, degrade to some degree to formproducts which cause adverse side effects such as diarrhea. For thisreason, many derivatives of naturally occurring macrolide antibioticssuch as erythromycin are esterified at the 6-position to minimizeformation of the degradation products following oral administration.

It would be advantageous to provide agents useful as antibiotics whichhave higher bioavailability, and are less subject to degradation andwhich have improved affinity for ribosomal RNA. The present inventionprovides such agents.

SUMMARY OF THE INVENTION

This invention is directed to novel multibinding compounds (agents) thatare macrolide antibiotics, aminoglycosides, lincosamides,oxazolidinones, streptogramins, tetracyclines or other compounds whichbind to ribosomal RNA and/or to one or more proteins involved inribosomal protein synthesis in the bacterium. The multibinding compoundsof this invention are useful as antibacterials, in particular, for grampositive, gram negative and anaerobic bacteria.

Accordingly, in one of its composition aspects, this invention providesa multibinding compound comprising from 2 to 10 ligands covalentlyattached to one or more linkers wherein each of said ligandsindependently comprises a macrolide antibiotic, aminoglycoside,lincosamide, oxazolidinone, streptogramin, tetracycline or othercompound which binds to ribosomal RNA and/or to one or more proteinsinvolved in ribosomal protein synthesis in the bacterium and adverselyaffects protein synthesis, and pharmaceutically-acceptable saltsthereof.

In another of its composition aspects, this invention provides amultibinding compound of formula I:(L)_(p)(X)_(q)  (I)

wherein each L is independently a ligand comprising a macrolideantibiotic, aminoglycoside, lincosamide, oxazolidinone, streptogramin,tetracycline or other compound which binds to ribosomal RNA and/or toone or more proteins involved in ribosomal protein synthesis in thebacterium; each X is independently a linker; p is an integer of from 2to 10; and q is an integer of from 1 to 20; andpharmaceutically-acceptable salts thereof.

Preferably, q is less than p in the multibinding compounds of thisinvention.

Preferably, each ligand, L, in the multibinding compound of formula I isindependently selected from the group consisting of erythromycin andester prodrugs/derivatives thereof, such as erythromycin stearate anderythromycin estolate; clarithromycin, roxythromycin, azithromycin,aureomycin, oleandomycin, sulfisoxazole, spiramycin, troleandomycin,josamycin, cytovaricin, linezolid, eperezolid, clindamycin [AntirobeR],lincomycin, quinupristin, dalfopristin (Synercid, Rhone-Poulenc Rorer),streptomycin, amikacin, gentamicin, kanamycin, neomycin, tobramycin,netilmicin, paromomycin, tetracycline, chlortetracycline, doxycycline,minocycline, declomycin, methacycline, spectinomycin, andoxytetracycline, and analogues thereof, which are well known to those ofskill in the art. Examples of suitable analogues include alkylated,esterified, amidated, alkoxylated, sulfonated, carboxylated,halogenated, phosphorylated, thiolated and hydroxylated analogues.

In still another of its composition aspects, this invention provides amultibinding compound of formula II:L′-X′-L′  II

wherein each L′ is independently a ligand comprising a macrolideantibiotic, aminoglycoside, lincosamide, oxazolidinone, streptogramin,tetracycline or other compound which binds to ribosomal RNA and/or toone or more proteins involved in ribosomal protein synthesis in thebacterium and X′ is a linker; and pharmaceutically-acceptable saltsthereof.

Preferably, in the multibinding compound of formula II, each ligand, L′,is independently selected from the group consisting of macrolideantibiotics, oxazolidinones, lincosamides, streptogramins, tetracyclinesand aminoglycosides, and X′ is a linker; and pharmaceutically-acceptablesalts thereof.

Preferably, in the above embodiments, each linker (i.e., X, X′ or X″)independently has the formula:—X^(a)-Z-(Y^(a)-Z)_(m)-Y^(b)-Z-X^(a)—

wherein

m is an integer of from 0 to 20;

X^(a) at each separate occurrence is selected from the group consistingof —O—, —S—, —NR—, —C(O)—, —C(O)O—, —C(O)NR—, —C(S), —C(S)O—, —C(S)NR—or a covalent bond where R is as defined below;

Z is at each separate occurrence is selected from the group consistingof alkylene, substituted alkylene, cycloalkylene, substitutedcylcoalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

Y^(a) and Y^(b) at each separate occurrence are selected from the groupconsisting of —C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —C(═NR′)—NR′—,—NR′—C(═NR′)—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—, —P(O)(OR′)—O—,—S(O)_(n)CR′R″—, —S(O)_(n)—NR′—, —S—S— and a covalent bond; where n is0, 1 or 2; and R, R′ and R″ at each separate occurrence are selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,aryl, heteroaryl and heterocyclic.

In yet another of its composition aspects, this invention provides apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an effective amount of a multibinding compound comprisingfrom 2 to 10 ligands covalently attached to one or more linkers whereineach of said ligands independently comprises a macrolide antibiotic,aminoglycoside, lincosamide, oxazolidinone, streptogramin, tetracyclineor other compound which binds to ribosomal RNA and/or to one or moreproteins involved in ribosomal protein synthesis in the bacterium, andpharmaceutically-acceptable salts thereof.

This invention is also directed to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and an effective amountof a multibinding compound of formula I or II.

The multibinding compounds of this invention are effective antibiotics.Accordingly, in one of its method aspects, this invention provides amethod for treating bacterial infections.

When used to treat bacterial infections, for example, the methodinvolves administering to a patient having a bacterial infection apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a therapeutically-effective amount of a multibindingcompound comprising from 2 to 10 ligands covalently attached to one ormore linkers wherein each of said ligands independently comprises amacrolide antibiotic, aminoglycoside, lincosamide, oxazolidinone,streptogramin, tetracycline or other compound which binds to ribosomalRNA and/or to one or more proteins involved in ribosomal proteinsynthesis in the bacterium; and pharmaceutically-acceptable saltsthereof.

This invention is also directed to general synthetic methods forgenerating large libraries of diverse multimeric compounds whichmultimeric compounds are candidates for possessing multibindingproperties with respect to ribosomal RNA and/or to one or more proteinsinvolved in ribosomal protein synthesis in the bacterium. The diversemultimeric compound libraries provided by this invention are synthesizedby combining a linker or linkers with a ligand or ligands to provide fora library of multimeric compounds wherein the linker and ligand eachhave complementary functional groups permitting covalent linkage. Thelibrary of linkers is preferably selected to have diverse propertiessuch as valency, linker length, linker geometry and rigidity,hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity andpolarization. The library of ligands is preferably selected to havediverse attachment points on the same ligand, different functionalgroups at the same site of otherwise the same ligand, and the like.

This invention is also directed to general synthetic methods forgenerating large libraries of diverse multimeric compounds whichmultimeric compounds are candidates for possessing multibindingproperties with respect to bacterial ribosomal RNA and/or to one or moreproteins involved in ribosomal protein synthesis in the bacterium. Thediverse multimeric compound libraries provided by this invention aresynthesized by combining a linker or linkers with a ligand or ligands toprovide for a library of multimeric compounds wherein the linker andligand each have complementary functional groups permitting covalentlinkage. The library of linkers is preferably selected to have diverseproperties such as valency, linker length, linker geometry and rigidity,hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity andpolarizability and/or polarization. The library of ligands is preferablyselected to have diverse attachment points on the same ligand, differentfunctional groups at the same site of otherwise the same ligand, and thelike.

This invention is also directed to libraries of diverse multimericcompounds which multimeric compounds are candidates for possessingmultibinding properties with respect to bacterial ribosomal RNA and/orto one or more proteins involved in ribosomal protein synthesis in thebacterium. These libraries are prepared via the methods described aboveand permit the rapid and efficient evaluation of what molecularconstraints impart multibinding properties to a ligand or a class ofligands targeting the bacterial ribosomal RNA and/or to one or moreproteins involved in ribosomal protein synthesis in the bacterium.

Accordingly, in one of its method aspects, this invention is directed toa method for identifying multimeric ligand compounds possessingmultibinding properties with respect to bacterial ribosomal RNA and/orto one or more proteins involved in ribosomal protein synthesis in thebacterium which method comprises:

(a) identifying a ligand or a mixture of ligands which bind to bacterialribosomal RNA and/or to one or more proteins involved in ribosomalprotein synthesis in the bacterium wherein each ligand contains at leastone reactive functionality;

(b) identifying a library of linkers wherein each linker in said librarycomprises at least two functional groups having complementary reactivityto at least one of the reactive functional groups of the ligand;

(c) preparing a multimeric ligand compound library by combining at leasttwo stoichiometric equivalents of the ligand or mixture of ligandsidentified in (a) with the library of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands;and

(d) assaying the multimeric ligand compounds produced in (c) above toidentify multimeric ligand compounds possessing multibinding properties.

In another of its method aspects, this invention is directed to a methodfor identifying multimeric ligand compounds possessing multibindingproperties which method comprises:

(a) identifying a library of ligands which bind to bacterial ribosomalRNA and/or to one or more proteins involved in ribosomal proteinsynthesis in the bacterium wherein each ligand contains at least onereactive functionality;

(b) identifying a linker or mixture of linkers wherein each linkercomprises at least two functional groups having complementary reactivityto at least one of the reactive functional groups of the ligand;

(c) preparing a multimeric ligand compound library by combining at leasttwo stoichiometric equivalents of the library of ligands identified in(a) with the linker or mixture of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands;and

(d) assaying the multimeric ligand compounds produced in (c) above toidentify multimeric ligand compounds possessing multibinding properties.

The preparation of the multimeric ligand compound library is achieved byeither the sequential or concurrent combination of the two or morestoichiometric equivalents of the ligands identified in (a) with thelinkers identified in (b). Sequential addition is preferred when amixture of different ligands is employed to ensure heteromeric ormultimeric compounds are prepared. Concurrent addition of the ligandsoccurs when at least a portion of the multimer comounds prepared arehomomultimeric compounds.

The assay protocols recited in (d) can be conducted on the multimericligand compound library produced in (c) above, or preferably, eachmember of the library is isolated by preparative liquid chromatographymass spectrometry (LCMS).

In one of its composition aspects, this invention is directed to alibrary of multimeric ligand compounds which may possess multivalentproperties which library is prepared by the method comprising:

(a) identifying a ligand or a mixture of ligands which bind to bacterialribosomal RNA and/or to one or more proteins involved in ribosomalprotein synthesis in the bacterium wherein each ligand contains at leastone reactive functionality;

(b) identifying a library of linkers wherein each linker in said librarycomprises at least two functional groups having complementary reactivityto at least one of the reactive functional groups of the ligand; and

(c) preparing a multimeric ligand compound library by combining at leasttwo stoichiometric equivalents of the ligand or mixture of ligandsidentified in (a) with the library of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands.

In another of its composition aspects, this invention is directed to alibrary of multimeric ligand compounds which bind to bacterial ribosomalRNA and/or to one or more proteins involved in ribosomal proteinsynthesis in the bacterium which may possess multivalent propertieswhich library is prepared by the method comprising:

(a) identifying a library of ligands which bind to bacterial ribosomalRNA and/or to one or more proteins involved in ribosomal proteinsynthesis in the bacterium wherein each ligand contains at least onereactive functionality;

(b) identifying a linker or mixture of linkers wherein each linkercomprises at least two functional groups having complementary reactivityto at least one of the reactive functional groups of the ligand; and

(c) preparing a multimeric ligand compound library by combining at leasttwo stoichiometric equivalents of the library of ligands identified in(a) with the linker or mixture of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands.

In a preferred embodiment, the library of linkers employed in either themethods or the library aspects of this invention is selected from thegroup comprising flexible linkers, rigid linkers, hydrophobic linkers,hydrophilic linkers, linkers of different geometry, acidic linkers,basic linkers, linkers of different polarizability and/or polarizationand amphiphilic linkers. For example, in one embodiment, each of thelinkers in the linker library may comprise linkers of different chainlength and/or having different complementary reactive groups. Suchlinker lengths can preferably range from about 2 to 100 Å.

In another preferred embodiment, the ligand or mixture of ligands isselected to have reactive functionality at different sites on theligands in order to provide for a range of orientations of said ligandon said multimeric ligand compounds. Such reactive functionalityincludes, by way of example, carboxylic acids, carboxylic acid halides,carboxyl esters, amines, halides, pseudohalides, isocyanates, vinylunsaturation, ketones, aldehydes, thiols, alcohols, anhydrides,boronates and precursors thereof It is understood, of course, that thereactive functionality on the ligand is selected to be complementary toat least one of the reactive groups on the linker so that a covalentlinkage can be formed between the linker and the ligand.

In other embodiments, the multimeric ligand compound is homomeric (i.e.,each of the ligands is the same, although it may be attached atdifferent points) or heteromeric (i.e., at least one of the ligands isdifferent from the other ligands).

In addition to the combinatorial methods described herein, thisinvention provides for an iterative process for rationally evaluatingwhat molecular constraints impart multibinding properties to a class ofmultimeric compounds or ligands targeting bacterial ribosomal RNA and/orto one or more proteins involved in ribosomal protein synthesis in thebacterium. Specifically, this method aspect is directed to a method foridentifying multimeric ligand compounds possessing multibindingproperties with respect to bacterial ribosomal RNA and/or to one or moreproteins involved in ribosomal protein synthesis in the bacterium whichmethod comprises:

(a) preparing a first collection or iteration of multimeric compoundswhich is prepared by contacting at least two stoichiometric equivalentsof the ligand or mixture of ligands which target bacterial ribosomal RNAand/or to one or more proteins involved in ribosomal protein synthesisin the bacterium with a linker or mixture of linkers wherein said ligandor mixture of ligands comprises at least one reactive functionality andsaid linker or mixture of linkers comprises at least two functionalgroups having complementary reactivity to at least one of the reactivefunctional groups of the ligand wherein said contacting is conductedunder conditions wherein the complementary functional groups react toform a covalent linkage between said linker and at least two of saidligands;

(b) assaying said first collection or iteration of multimeric compoundsto assess which if any of said multimeric compounds possess multibindingproperties;

(c) repeating the process of (a) and (b) above until at least onemultimeric compound is found to possess multibinding properties;

(d) evaluating what molecular constraints imparted multibindingproperties to the multimeric compound or compounds found in the firstiteration recited in (a)–(c) above;

(e) creating a second collection or iteration of multimeric compoundswhich elaborates upon the particular molecular constraints impartingmultibinding properties to the multimeric compound or compounds found insaid first iteration;

(f) evaluating what molecular constraints imparted enhanced multibindingproperties to the multimeric compound or compounds found in the secondcollection or iteration recited in (e) above;

(g) optionally repeating steps (e) and (f) to further elaborate uponsaid molecular constraints.

Preferably, steps (e) and (f) are repeated at least two times, morepreferably at from 2–50 times, even more preferably from 3 to 50 times,and still more preferably at least 5–50 times.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of representative macrolideantibiotics, aminoglycosides and lincosamides, oxazolidinones,streptogramins, tetracycline which can be used to prepare the compoundsof the present invention.

FIGS. 2–20 are schematic illustrations of the preparation of compoundsof formula I–XIX, respectively.

FIG. 21 is a schematic illustration of the preparation of compounds offormulas XXa, XXb and XXc.

FIGS. 22–24, 24 a, 25, 25 a, and 26–37 are schematic illustrations ofthe preparation of compounds of formula XXI–XXXVIII, respectively.

FIG. 38 is a schematic illustration of the preparation of compounds offormulas XXXIX-A and XXXIX-B.

FIGS. 39–49 are schematic illustrations of the preparation of compoundsof formula XL-L.

FIG. 50 is a schematic illustration of the preparation of compounds offormulas LI-A and LI-B.

FIG. 51 is a schematic illustration of the preparation of compounds offormulas LII-A and LII-B.

FIG. 52 is a schematic illustration of the preparation of compounds offormula LIII-A and LIII-B.

FIGS. 53–55 are schematic illustrations of the preparation of compoundsof formula LIV–LVI.

FIG. 56 illustrates examples of multibinding compounds comprising 2ligands attached in different forms to a linker.

FIG. 57 illustrates examples of multibinding compounds comprising 3ligands attached in different forms to a linker.

FIG. 58 illustrates examples of multibinding compounds comprising 4ligands attached in different forms to a linker.

FIG. 59 illustrates examples of multibinding compounds comprisingbetween 5 and 10 ligands attached in different forms to a linker.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to multibinding compounds which are macrolideantibiotics, aminoglycosides, lincosamides, oxazolidinones,streptogramins, tetracyclines or other compounds which bind to bacterialribosomal RNA and/or to one or more proteins involved in ribosomalprotein synthesis in the bacterium, preferably in a manner whichinhibits protein expression and results in an antibacterial activity,pharmaceutical compositions containing such compounds and methods ofantibacterial treatment. When discussing such compounds, compositions ormethods, the following terms have the following meanings unlessotherwise indicated. Any undefined terms have their art recognizedmeanings.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from 1 to 40 carbon atoms,more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6carbon atoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl,and the like.

The term “substituted alkyl” refers to an alkyl group as defined above,having from 1 to 5 substituents, and preferably 1 to 3 substituents,selected from the group consisting of alkoxy, substituted alkoxy,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl. —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

The term “alkylene” refers to a diradical of a branched or unbranchedsaturated hydrocarbon chain, preferably having from 1 to 40 carbonatoms, more preferably 1 to 10 carbon atoms and even more preferably 1to 6 carbon atoms. This term is exemplified by groups such as methylene(—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers (e.g., —CH₂CH₂CH₂—and —CH(CH₃)CH₂—) and the like.

The term “substituted alkylene” refers to an alkylene group, as definedabove, having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl. Additionally, such substituted alkylene groupsinclude those where 2 substituents on the alkylene group are fused toform one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fusedto the alkylene group. Preferably such fused groups contain from 1 to 3fused ring structures.

The term “alkaryl” refers to the groups -alkylene-aryl and -substitutedalkylene-aryl where alkylene, substituted alkylene and aryl are definedherein. Such alkaryl groups are exemplified by benzyl, phenethyl and thelike.

The term “alkoxy” refers to the groups alkyl-O—, alkenyl-O—,cycloalkyl-O—, cycloalkenyl-O—, and alkynyl-O—, where alkyl, alkenyl,cycloalkyl, cycloalkenyl, and alkynyl are as defined herein. Preferredalkoxy groups are alkyl-O— and include, by way of example, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O-substituted alkyl, substituted alkylene-O-alkyl andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.Preferred alkylalkoxy groups are alkylene-O-alkyl and include, by way ofexample, methylenemethoxy (—CH₂OCH₃), ethylenemethoxy (—CH₂CH₂OCH₃),n-propylene-iso-propoxy (—CH₂CH₂CH₂OCH(CH₃)₂), methylene-t-butoxy(—CH₂—O—C(CH₃)₃) and the like.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.Preferred alkylthioalkoxy groups are alkylene-S-alkyl and include, byway of example, methylenethiomethoxy (—CH₂SCH₃), ethylenethiomethoxy(—CH₂CH₂SCH₃), n-propylene-iso-thiopropoxy (—CH₂CH₂CH₂SCH(CH₃)₂),methylene-t-thiobutoxy (—CH₂SC(CH₃)₃) and the like.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group preferably having from 2 to 40 carbonatoms, more preferably 2 to 10 carbon atoms and even more preferably 2to 6 carbon atoms and having at least 1 and preferably from 1–6 sites ofvinyl unsaturation. Preferred alkenyl groups include ethenyl (—CH═CH₂),n-propenyl (—CH₂CH═CH₂), iso-propenyl (—C(CH₃)═CH₂), and the like.

The term “substituted alkenyl” refers to an alkenyl group as definedabove having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

The term “alkenylene” refers to a diradical of a branched or unbranchedunsaturated hydrocarbon group preferably having from 2 to 40 carbonatoms, more preferably 2 to 10 carbon atoms and even more preferably 2to 6 carbon atoms and having at least 1 and preferably from 1–6 sites ofvinyl unsaturation. This term is exemplified by groups such asethenylene (—CH═CH—), the propenylene isomers (e.g., —CH₂CH═CH— and—C(CH₃)═CH—) and the like.

The term “substituted alkenylene” refers to an alkenylene group asdefined above having from 1 to 5 substituents, and preferably from 1 to3 substituents, selected from the group consisting of alkoxy,substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substitutedamino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen,hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Additionally,such substituted alkenylene groups include those where 2 substituents onthe alkenylene group are fused to form one or more cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,heterocyclic or heteroaryl groups fused to the alkenylene group.

The term “alkynyl” refers to a monoradical of an unsaturated hydrocarbonpreferably having from 2 to 40 carbon atoms, more preferably 2 to 20carbon atoms and even more preferably 2 to 6 carbon atoms and having atleast 1 and preferably from 1–6 sites of acetylene (triple bond)unsaturation. Preferred alkynyl groups include ethynyl (—C≡CH),propargyl (—CH₂C≡CH) and the like.

The term “substituted alkynyl” refers to an alkynyl group as definedabove having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

The term “alkynylene” refers to a diradical of an unsaturatedhydrocarbon preferably having from 2 to 40 carbon atoms, more preferably2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms andhaving at least 1 and preferably from 1–6 sites of acetylene (triplebond) unsaturation. Preferred alkynylene groups include ethynylene(—C≡C—), propargylene (—CH₂C≡C—) and the like.

The term “substituted alkynylene” refers to an alkynylene group asdefined above having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl

The term “acyl” refers to the groups HC(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, cycloalkyl—C(O)—, substituted cycloalkyl-C(O)—,cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—, aryl-C(O)—,heteroaryl-C(O)— and heterocyclic-C(O)— where alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “acylamino” or “aminocarbonyl” refers to the group —C(O)NRRwhere each R is independently hydrogen, alkyl, substituted alkyl, aryl,heteroaryl, heterocyclic or where both R groups are joined to form aheterocyclic group (e.g., morpholino) wherein alkyl, substituted alkyl,aryl, heteroaryl and heterocyclic are as defined herein.

The term “aminoacyl” refers to the group —NRC(O)R where each R isindependently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, orheterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

The term “aminoacyloxy” or “alkoxycarbonylamino” refers to the group—NRC(O)OR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclic-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclic are as defined herein.

The term “aryl” refers to an unsaturated aromatic carbocyclic group offrom 6 to 20 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed (fused) rings (e.g., naphthyl or anthryl). Preferredaryls include phenyl, naphthyl and the like.

Unless otherwise constrained by the definition for the aryl substituent,such aryl groups can optionally be substituted with from 1 to 5substituents, preferably 1 to 3 substituents, selected from the groupconsisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substitutedalkoxy, substituted alkenyl, substituted alkynyl, substitutedcycloalkyl, substituted cycloalkenyl, amino, substituted amino,aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy.

The term “aryloxy” refers to the group aryl-O— wherein the aryl group isas defined above including optionally substituted aryl groups as alsodefined above.

The term “arylene” refers to the diradical derived from aryl (includingsubstituted aryl) as defined above and is exemplified by 1,2-phenylene,1,3-phenylene, 1,4-phenylene, 1,2-naphthylene and the like.

The term “amino” refers to the group —NH₂.

The term “substituted amino refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl and heterocyclic provided thatboth R's are not hydrogen.

The term “carboxyalkyl” or “alkoxycarbonyl” refers to the groups“—C(O)O-alkyl”, “—C(O)O-substituted alkyl”, “—C(O)O-cycloalkyl”,“—C(O)O-substituted cycloalkyl”, “—C(O)O-alkenyl”, “—C(O)O-substitutedalkenyl”, “—C(O)O-alkynyl” and “—C(O)O-substituted alkynyl” where alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, alkynyl and substituted alkynyl alkynyl are asdefined herein.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1to 5 substituents, and preferably 1 to 3 substituents, selectedfrom the group consisting of alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 20carbon atoms having a single cyclic ring and at least one point ofinternal unsaturation. Examples of suitable cycloalkenyl groups include,for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and thelike.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, and preferably 1 to 3 substituents, selectedfrom the group consisting of alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

The term “heteroaryl” refers to an aromatic group of from 1 to 15 carbonatoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfurwithin at least one ring (if there is more than one ring).

Unless otherwise constrained by the definition for the heteroarylsubstituent, such heteroaryl groups can be optionally substituted with 1to 5 substituents, preferably 1 to 3 substituents, selected from thegroup consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a singlering (e.g., pyridyl or furyl) or multiple condensed rings (e.g.,indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl,pyrrolyl and furyl.

The term “heteroaryloxy” refers to the group heteroaryl-O—.

The term “heteroarylene” refers to the diradical group derived fromheteroaryl (including substituted heteroaryl), as defined above, and isexemplified by the groups 2,6-pyridylene, 2,4-pyridiylene,1,2-quinolinylene, 1,8-quinolinylene, 1,4-benzofuranylene,2,5-pyridnylene, 2,5-indolenyl and the like.

The term “heterocycle” or “heterocyclic” refers to a monoradicalsaturated or unsaturated group having a single ring or multiplecondensed rings, from 1 to 40 carbon atoms and from 1 to 10 heteroatoms, preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen within the ring.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, and preferably 1 to 3 substituents, selected from the groupconsisting of alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Suchheterocyclic groups can have a single ring or multiple condensed rings.Preferred heterocyclics include morpholino, piperidinyl, and the like.

Examples of nitrogen heterocycles and heteroaryls include, but are notlimited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, indoline, morpholino, piperidinyl,tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containingheterocycles.

The term “heterocyclooxy” refers to the group heterocyclic-O—.

The term “thioheterocyclooxy” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein, and is exemplified by the groups2,6-morpholino, 2,5-morpholino and the like.

The term “oxyacylamino” or “aminocarbonyloxy” refers to the group—OC(O)NRR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “spiro-attached cycloalkyl group” refers to a cycloalkyl groupattached to another ring via one carbon atom common to both rings.

The term “thiol” refers to the group —SH.

The term “thioalkoxy” refers to the group —S-alkyl.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined above including optionally substituted aryl groupsalso defined above.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined above including optionallysubstituted aryl groups as also defined above.

As to any of the above groups which contain one or more substituents, itis understood, of course, that such groups do not contain anysubstitution or substitution patterns which are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds, whether the isomers are those arisingin the ligands, the linkers, or the multivalent constructs including theligands and linkers.

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the multibindingcompounds of this invention and which are not biologically or otherwiseundesirable. In many cases, the multibinding compounds of this inventionare capable of forming acid and/or base salts by virtue of the presenceof amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group.

Examples of suitable amines include, by way of example only,isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine,tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine,purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and thelike. It should also be understood that other carboxylic acidderivatives would be useful in the practice of this invention, forexample, carboxylic acid amides, including carboxamides, lower alkylcarboxamides, dialkyl carboxamides, and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

The term “pharmaceutically-acceptable cation” refers to the cation of apharmaceutically-acceptable salt.

The term “protecting group” or “blocking group” refers to any groupwhich when bound to one or more hydroxyl, thiol, amino or carboxylgroups of the compounds (including intermediates thereof) preventsreactions from occurring at these groups and which protecting group canbe removed by conventional chemical or enzymatic steps to reestablishthe hydroxyl, thiol, amino or carboxyl group. The particular removableblocking group employed is not critical and preferred removable hydroxylblocking groups include conventional substituents such as allyl, benzyl,acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl,t-butyl-diphenylsilyl and any other group that can be introducedchemically onto a hydroxyl functionality and later selectively removedeither by chemical or enzymatic methods in mild conditions compatiblewith the nature of the product.

Preferred removable thiol blocking groups include disulfide groups, acylgroups, benzyl groups, and the like.

Preferred removable amino blocking groups include conventionalsubstituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ),fluotenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC), and the likewhich can be removed by conventional conditions compatible with thenature of the product.

Preferred carboxyl protecting groups include esters such as methyl,ethyl, propyl, t-butyl etc. which can be removed by mild conditionscompatible with the nature of the product.

The term “optional” or “optionally” means that the subsequentlydescribed event, circumstance or substituent may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

The term “ligand” as used herein denotes a compound that binds toribosomal RNA and/or to one or more proteins involved in ribosomalprotein synthesis in the bacterium. The specific region or regions ofthe ligand that is (are) recognized by the enzyme is designated as the“ligand domain”. A ligand may be either capable of binding to an enzymeby itself, or may require the presence of one or more non-ligandcomponents for binding (e.g., Ca⁺², Mg⁺² or a water molecule is requiredfor the binding of a ligand to various ligand binding sites).

Examples of ligands useful in this invention are described herein.General classes of compounds useful as ligands include macrolideantibiotics, oxazolidinones, lincosamides, streptogramins, tetracyclinesand aminoglycosides. Examples of macrolide antibiotics includeerythromycin and ester derivatives thereof, such as erythromycinstearate and erythromycin estolate; clarithromycin, roxythromycin,azithromycin, streptomycin, aureomycin, oleandomycin, sulfisoxazole,spiramycin, troleandomycin, josamycin and cytovaricin. Examples ofoxazolidinones include linezolid and eperezolid. Examples oflincosamides include clindamycin [AntirobeR] and lincomycin. Examples ofstreptogramins include quinupristin and dalfopristin (Synercid,Rhone-Poulenc Rorer). Examples of aminoglycosides include streptomycin,amikacin, gentamicin, kanamycin, neomycin, tobramycin, netilmicin andparomomycin. Examples of tetracyclines include tetracycline,chlortetracycline, doxycycline, minocycline, declomycin, methacyclineand oxytetracycline.

Those skilled in the art will appreciate that portions of the ligandstructure that are not essential for specific molecular recognition andbinding-activity may be varied substantially, replaced or substitutedwith unrelated structures (for example, with ancillary groups as definedbelow) and, in some cases, omitted entirely without affecting thebinding interaction. The primary requirement for a ligand is that it hasa ligand domain as defined above. It is understood that the term ligandis not intended to be limited to compounds known to be useful in bindingto bacterial ribosomal RNA and/or to one or more proteins involved inribosomal protein synthesis in the bacterium (e.g., known drugs). Thoseskilled in the art will understand that the term ligand can equallyapply to a molecule that is not normally associated with bacterialribosomal RNA binding properties. In addition, it should be noted thatligands that exhibit marginal activity or lack useful activity asmonomers can be highly active as multivalent compounds because of thebenefits conferred by multivalency.

The term “multibinding compound or agent” refers to a compound that iscapable of multivalency, as defined below, and which has 2–10 ligandscovalently bound to one or more linkers which may be the same ordifferent. Multibinding compounds provide a biological and/ortherapeutic effect greater than the aggregate of unlinked ligandsequivalent thereto which are made available for binding. That is to saythat the biological and/or therapeutic effect of the ligands attached tothe multibinding compound is greater than that achieved by the sameamount of unlinked ligands made available for binding to the ligandbinding sites (receptors). The phrase “increased biological ortherapeutic effect” includes, for example: increased affinity, increasedselectivity for target, increased specificity for target, increasedpotency, increased efficacy, decreased toxicity, improved duration ofactivity or action, decreased side effects, increased therapeutic index,improved bioavailability, improved pharmacokinetics, improved activityspectrum, and the like. The multibinding compounds of this inventionwill exhibit at least one and preferably more than one of theabove-mentioned effects.

The term “potency” refers to the minimum concentration at which a ligandis able to achieve a desirable biological or therapeutic effect. Thepotency of a ligand is typically proportional to its affinity for itsligand binding site. In some cases, the potency may be non-linearlycorrelated with its affinity. In comparing the potency of two drugs,e.g., a multibinding agent and the aggregate of its unlinked ligand, thedose-response curve of each is determined under identical testconditions (e.g., in an in vitro or in vivo assay, in an appropriateanimal model). The finding that the multibinding agent produces anequivalent biological or therapeutic effect at a lower concentrationthan the aggregate unlinked ligand is indicative of enhanced potency.

The term “univalency” as used herein refers to a single bindinginteraction between one ligand as defined herein with one ligand bindingsite as defined herein. It should be noted that a compound havingmultiple copies of a ligand (or ligands) exhibit univalency when onlyone ligand is interacting with a ligand binding site. Examples ofunivalent interactions are depicted below.

The term “multivalency” as used herein refers to the concurrent bindingof from 2 to 10 linked ligands (which may be the same or different) andtwo or more corresponding receptors (ligand binding sites) on one ormore bacterial ribosomes which may be the same or different.

For example, two ligands connected through a linker that bindconcurrently to two ligand binding sites would be considered asbivalency; three ligands thus connected would be an example oftrivalency. An example of trivalent binding, illustrating a multibindingcompound bearing three ligands versus a monovalent binding interaction,is shown below:

It should be understood that all compounds that contain multiple copiesof a ligand attached to a linker or to linkers do not necessarilyexhibit the phenomena of multivalency, i.e., that the biological and/ortherapeutic effect of the multibinding agent is greater than the sum ofthe aggregate of unlinked ligands made available for binding to theligand binding site (receptor). For multivalency to occur, the ligandsthat are connected by a linker or linkers have to be presented to theirligand binding sites by the linker(s) in a specific manner in order tobring about the desired ligand-orienting result, and thus produce amultibinding event.

The term “selectivity” or “specificity” is a measure of the bindingpreferences of a ligand for different ligand binding sites (receptors).The selectivity of a ligand with respect to its target ligand bindingsite relative to another ligand binding site is given by the ratio ofthe respective values of K_(d) (i.e., the dissociation constants foreach ligand-receptor complex) or, in cases where a biological effect isobserved below the K_(d), the ratio of the respective EC₅₀'s (i.e., theconcentrations that produce 50% of the maximum response for the ligandinteracting with the two distinct ligand binding sites (receptors)).

The term “ligand binding site” denotes the site on the bacterialribosomal RNA, which may be specific RNA and/or a site on one or more ofthe proteins involved in ribosomal protein synthesis in the bacteriumthat recognizes a ligand domain and provides a binding partner for theligand. The ligand binding site may be defined by monomeric ormultimeric structures.

It should be recognized that the ligand binding sites of the ribosomethat participate in biological multivalent binding interactions areconstrained to varying degrees by their intra- and inter-molecularassociations (e.g., such macromolecular structures may be covalentlyjoined to a single structure, noncovalently associated in a multimericstructure, embedded in a membrane or polymeric matrix, and so on) andtherefore have less translational and rotational freedom than if thesame structures were present as monomers in solution.

The term “inert organic solvent” means a solvent which is inert underthe conditions of the reaction being described in conjunction therewithincluding, by way of example only, benzene, toluene, acetonitrile,tetrahydrofuran, dimethylformamide, chloroform, methylene chloride,diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol,ethanol, propanol, isopropanol, t-butanol, dioxane, pyridine, and thelike. Unless specified to the contrary, the solvents used in thereactions described herein are inert solvents.

The term “treatment” refers to any treatment of a pathologic conditionin a mammal, particularly a human, and includes:

(i) preventing the pathologic condition from occurring in a subjectwhich may be predisposed to the condition but has not yet been diagnosedwith the condition and, accordingly, the treatment constitutesprophylactic treatment for the disease condition;

(ii) inhibiting the pathologic condition, i.e., arresting itsdevelopment;

(iii) relieving the pathologic condition, i.e., causing regression ofthe pathologic condition; or

(iv) relieving the conditions mediated by the pathologic condition.

The term “pathologic condition which is modulated by treatment with aligand” covers all disease states (i.e., pathologic conditions) whichare generally acknowledged in the art to be usefully treated with aligand for the bacterial ribosomal RNA and/or for one or more proteinsinvolved in ribosomal protein synthesis in the bacterium, in general,and those disease states which have been found to be usefully treated bya specific multibinding compound of our invention. Such disease statesinclude, by way of example only, the treatment of bacterial infections.

Examples of bacteria which can be treated with the compositions andmethods described herein include gram-positive cocci, for example,staphylococci and streptococci, gram-positive rods such ascorynebacteria and diphtheroids, bacilli, mycobacteria and Nocardia,gram-negative rods such as enteric gram-negative rods, Pseudomonas,curved gram-negative rods, parvobacteria and Haemophilus, gram-negativecocci such as Neisseria, obligate anaerobes such as Clostridium,non-sporing anaerobes, and unusual bacteria such as spirochaetes,rickettsia and chlamydia. Specific examples of bacterial diseasesinclude chlamydia, gonorrhea, salmonellosis, shigellosis, tuberculosis,syphilis, bacterial pneumonia, bacterial sepsis, urinary tractinfections, bacterial upper respiratory tract infections, otitis media,and lyme disease.

While not wishing to be bound to a particular theory, it is believedthat the compounds described herein inhibit growth of bacteria bybinding to their 16S or 23S ribosomal subunits, or to one or moreproteins present on the bacterial ribosome, and inhibiting proteinsynthesis.

The term “therapeutically effective amount” refers to that amount ofmultibinding compound which is sufficient to effect treatment, asdefined above, when administered to a mammal in need of such treatment.The therapeutically effective amount will vary depending upon thesubject and disease condition being treated, the weight and age of thesubject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art.

The term “linker”, identified where appropriate by the symbol X, X′ orX″, refers to a group or groups that covalently links from 2 to 10ligands (as identified above) in a manner that provides for a compoundcapable of multivalency. Among other features, the linker is aligand-orienting entity that permits attachment of multiple copies of aligand (which may be the same or different) thereto. In some cases, thelinker may itself be biologically active. The term “linker” does not,however, extend to cover solid inert supports such as beads, glassparticles, fibers, and the like. But it is understood that themultibinding compounds of this invention can be attached to a solidsupport if desired. For example, such attachment to solid supports canbe made for use in separation and purification processes and similarapplications.

The term “library” refers to at least 3, preferably from 10² to 10⁹ andmore preferably from 10² to 10⁴ multimeric compounds. Preferably, thesecompounds are prepared as a multiplicity of compounds in a singlesolution or reaction mixture which permits facile synthesis thereof. Inone embodiment, the library of multimeric compounds can be directlyassayed for multibinding properties. In another embodiment, each memberof the library of multimeric compounds is first isolated and,optionally, characterized. This member is then assayed for multibindingproperties.

The term “collection” refers to a set of multimeric compounds which areprepared either sequentially or concurrently (e.g., combinatorially).The collection comprises at least 2 members; preferably from 2 to 10⁹members and still more preferably from 10 to 10⁴ members.

The term “multimeric compound” refers to compounds comprising from 2 to10 ligands covalently connected through at least one linker whichcompounds may or may not possess multibinding properties (as definedherein).

The term “pseudohalide” refers to functional groups which react indisplacement reactions in a manner similar to a halogen. Such functionalgroups include, by way of example, mesyl, tosyl, azido and cyano groups.

The extent to which multivalent binding is realized depends upon theefficiency with which the linker or linkers that joins the ligandspresents these ligands to the array of available ligand binding sites.Beyond presenting these ligands for multivalent interactions with ligandbinding sites, the linker or linkers spatially constrains theseinteractions to occur within dimensions defined by the linker orlinkers. Thus, the structural features of the linker (valency, geometry,orientation, size, flexibility, chemical composition, etc.) are featuresof multibinding agents that play an important role in determining theiractivities.

The linkers used in this invention are selected to allow multivalentbinding of ligands to the ligand binding sites of bacterial ribosomalRNA and/or of one or more proteins involved in ribosomal proteinsynthesis in the bacterium, wherever such sites are located on thereceptor structure.

The ligands are covalently attached to the linker or linkers usingconventional chemical techniques providing for covalent linkage of theligand to the linker or linkers. Reaction chemistries resulting in suchlinkages are well known in the art and involve the use of complementaryfunctional groups on the linker and ligand. Preferably, thecomplementary functional groups on the linker are selected relative tothe functional groups available on the ligand for bonding or which canbe introduced onto the ligand for bonding. Again, such complementaryfunctional groups are well known in the art. For example, reactionbetween a carboxylic acid of either the linker or the ligand and aprimary or secondary amine of the ligand or the linker in the presenceof suitable, well-known activating agents results in formation of anamide bond covalently linking the ligand to the linker; reaction betweenan amine group of either the linker or the ligand and a sulfonyl halideof the ligand or the linker results in formation of a sulfonamide bondcovalently linking the ligand to the linker; and reaction between analcohol or phenol group of either the linker or the ligand and an alkylor aryl halide of the ligand or the linker results in formation of anether bond covalently linking the ligand to the linker.

Table I below illustrates numerous complementary reactive groups and theresulting bonds formed by reaction therebetween.

TABLE I Representative Complementary Binding Chemistries First ReactiveGroup Second Reactive Group Linkage hydroxyl isocyanate urethane amineepoxide β-hydroxyamine sulfonyl halide amine sulfonamide carboxyl amineamide hydroxyl alkyl/aryl halide ether aldehyde amine/NaCNBH₄ amineketone amine/NaCNBH₄ amine amine isocyanate urea

The linker is attached to the ligand at a position that retains liganddomain-ligand binding site interaction and specifically which permitsthe ligand domain of the ligand to orient itself to bind to the ligandbinding site. Such positions and synthetic protocols for linkage arewell known in the art. The term linker embraces everything that is notconsidered to be part of the ligand.

The relative orientation in which the ligand domains are displayedderives from the particular point or points of attachment of the ligandsto the linker, and on the framework geometry. The determination of whereacceptable substitutions can be made on a ligand is typically based onprior knowledge of structure-activity relationships (SAR) of the ligandand/or congeners and/or structural information about ligand-receptorcomplexes (e.g., X-ray crystallography, NMR, and the like). Suchpositions and the synthetic methods for covalent attachment are wellknown in the art. Following attachment to the selected linker (orattachment to a significant portion of the linker, for example 2–10atoms of the linker), the univalent linker-ligand conjugate may betested for retention of activity in the relevant assay.

Suitable linkers and ligands are discussed more fully below.

At present, it is preferred that the multibinding agent is a bivalentcompound, e.g., two ligands which are covalently linked to linker X.

Methodology

The linker, when covalently attached to multiple copies of the ligands,provides a biocompatible, substantially non-immunogenic multibindingcompound. The biological activity of the multibinding compound is highlysensitive to the valency, geometry, composition, size, flexibility orrigidity, etc. of the linker and, in turn, on the overall structure ofthe multibinding compound, as well as the presence or absence of anionicor cationic charge, the relative hydrophobicity/hydrophilicity of thelinker, and the like on the linker. Accordingly, the linker ispreferably chosen to maximize the biological activity of themultibinding compound. The linker may be chosen to enhance thebiological activity of the molecule. In general, the linker may bechosen from any organic molecule construct that orients two or moreligands to their ligand binding sites to permit multivalency. In thisregard, the linker can be considered as a “framework” on which theligands are arranged in order to bring about the desiredligand-orienting result, and thus produce a multibinding compound.

For example, different orientations can be achieved by including in theframework groups containing mono- or polycyclic groups, including aryland/or heteroaryl groups, or structures incorporating one or morecarbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylenegroups). Other groups can also include oligomers and polymers which arebranched- or straight-chain species. In preferred embodiments, rigidityis imparted by the presence of cyclic groups (e.g., aryl, heteroaryl,cycloalkyl, heterocyclic, etc.). In other preferred embodiments, thering is a six or ten member ring. In still further preferredembodiments, the ring is an aromatic ring such as, for example, phenylor naphthyl.

Different hydrophobic/hydrophilic characteristics of the linker as wellas the presence or absence of charged moieties can readily be controlledby the skilled artisan. For example, the hydrophobic nature of a linkerderived from hexamethylene diamine (H₂N(CH₂)₆NH₂) or related polyaminescan be modified to be substantially more hydrophilic by replacing thealkylene group with a poly(oxyalkylene) group such as found in thecommercially available “Jeffamines”. By controlling thehydrophilicity/hydrophobicity, the ability of the compounds to cross theblood/brain barrier can be controlled. This can be important when onewishes to maximize or minimize CNS effects.

Examples of molecular structures in which the above bonding patternscould be employed as components of the linker are shown below.

The identification of an appropriate framework geometry and size forligand domain presentation are important steps in the construction of amultibinding compound with enhanced activity. Systematic spatialsearching strategies can be used to aid in the identification ofpreferred frameworks through an iterative process. Numerous strategiesare known to those skilled in the art of molecular design and can beused for preparing compounds of this invention.

It is to be noted that core structures other than those shown here canbe used for determining the optimal framework display orientation of theligands. The process may require the use of multiple copies of the samecentral core structure or combinations of different types of displaycores.

The above-described process can be extended to trimers and compounds ofhigher valency.

Assays of each of the individual compounds of a collection generated asdescribed above will lead to a subset of compounds with the desiredenhanced activities (e.g., potency, selectivity, etc.). The analysis ofthis subset using a technique such as Ensemble Molecular Dynamics willprovide a framework orientation that favors the properties desired. Awide diversity of linkers is commercially available (see, e.g.,Available Chemical Directory (ACD)). Many of the linkers that aresuitable for use in this invention fall into this category. Other can bereadily synthesized by methods well known in the art and/or aredescribed below.

Having selected a preferred framework geometry, the physical propertiesof the linker can be optimized by varying the chemical compositionthereof. The composition of the linker can be varied in numerous ways toachieve the desired physical properties for the multibinding compound.

It can therefore be seen that there is a plethora of possibilities forthe composition of a linker. Examples of linkers include aliphaticmoieties, aromatic moieties, steroidal moieties, peptides, and the like.Specific examples are peptides or polyamides, hydrocarbons, aromaticgroups, ethers, lipids, cationic or anionic groups, or a combinationthereof.

Examples are given below, but it should be understood that variouschanges may be made and equivalents may be substituted without departingfrom the true spirit and scope of the invention. For example, propertiesof the linker can be modified by the addition or insertion of ancillarygroups into or onto the linker, for example, to change the solubility ofthe multibinding compound (in water, fats, lipids, biological fluids,etc.), hydrophobicity, hydrophilicity, linker flexibility, antigenicity,stability, and the like. For example, the introduction of one or morepoly(ethylene glycol) (PEG) groups onto or into the linker enhances thehydrophilicity and water solubility of the multibinding compound,increases both molecular weight and molecular size and, depending on thenature of the unPEGylated linker, may increase the in vivo retentiontime. Further PEG may decrease antigenicity and potentially enhances theoverall rigidity of the linker.

Ancillary groups which enhance the water solubility/hydrophilicity ofthe linker and, accordingly, the resulting multibinding compounds areuseful in practicing this invention. Thus, it is within the scope of thepresent invention to use ancillary groups such as, for example, smallrepeating units of ethylene glycols, alcohols, polyols (e.g., glycerin,glycerol propoxylate, saccharides, including mono-, oligosaccharides,etc.), carboxylates (e.g., small repeating units of glutamic acid,acrylic acid, etc.), amines (e.g., tetraethylenepentamine), and thelike) to enhance the water solubility and/or hydrophilicity of themultibinding compounds of this invention. In preferred embodiments, theancillary group used to improve water solubility/hydrophilicity will bea polyether.

The incorporation of lipophilic ancillary groups within the structure ofthe linker to enhance the lipophilicity and/or hydrophobicity of themultibinding compounds described herein is also within the scope of thisinvention. Lipophilic groups useful with the linkers of this inventioninclude, by way of example only, aryl and heteroaryl groups which, asabove, may be either unsubstituted or substituted with other groups, butare at least substituted with a group which allows their covalentattachment to the linker. Other lipophilic groups useful with thelinkers of this invention include fatty acid derivatives which do notform bilayers in aqueous medium until higher concentrations are reached.

Also within the scope of this invention is the use of ancillary groupswhich result in the multibinding compound being incorporated or anchoredinto a vesicle or other membranous structure such as a liposome or amicelle. The term “lipid” refers to any fatty acid derivative that iscapable of forming a bilayer or a micelle such that a hydrophobicportion of the lipid material orients toward the bilayer while ahydrophilic portion orients toward the aqueous phase. Hydrophiliccharacteristics derive from the presence of phosphato, carboxylic,sulfato, amino, sulfhydryl, nitro and other like groups well known inthe art. Hydrophobicity could be conferred by the inclusion of groupsthat include, but are not limited to, long chain saturated andunsaturated aliphatic hydrocarbon groups of up to 20 carbon atoms andsuch groups substituted by one or more aryl, heteroaryl, cycloalkyl,and/or heterocyclic group(s). Preferred lipids are phosphoglycerides andsphingolipids, representative examples of which includephosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine ordilinoleoylphosphatidylcholine could be used. Other compounds lackingphosphorus, such as sphingolipid and glycosphingolipid families are alsowithin the group designated as lipid. Additionally, the amphipathiclipids described above may be mixed with other lipids includingtriglycerides and sterols.

The flexibility of the linker can be manipulated by the inclusion ofancillary groups which are bulky and/or rigid. The presence of bulky orrigid groups can hinder free rotation about bonds in the linker or bondsbetween the linker and the ancillary group(s) or bonds between thelinker and the functional groups. Rigid groups can include, for example,those groups whose conformational lability is restrained by the presenceof rings and/or multiple bonds within the group, for example, aryl,heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups. Othergroups which can impart rigidity include polypeptide groups such asoligo- or polyproline chains.

Rigidity may also be imparted by internal hydrogen bonding or byhydrophobic collapse.

Bulky groups can include, for example, large atoms, ions (e.g., iodine,sulfur, metal ions, etc.) or groups containing large atoms, polycyclicgroups, including aromatic groups, non-aromatic groups and structuresincorporating one or more carbon-carbon multiple bonds (i.e., alkenesand alkynes). Bulky groups can also include oligomers and polymers whichare branched- or straight-chain species. Species that are branched areexpected to increase the rigidity of the structure more per unitmolecular weight gain than are straight-chain species.

In preferred embodiments, rigidity is imparted by the presence of cyclicgroups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.). Inother preferred embodiments, the linker comprises one or moresix-membered rings. In still further preferred embodiments, the ring isan aryl group such as, for example, phenyl or naphthyl.

Rigidity can also be imparted electrostatically. Thus, if the ancillarygroups are either positively or negatively charged, the similarlycharged ancillary groups will force the presenter linker into aconfiguration affording the maximum distance between each of the likecharges. The energetic cost of bringing the like-charged groups closerto each other will tend to hold the linker in a configuration thatmaintains the separation between the like-charged ancillary groups.Further ancillary groups bearing opposite charges will tend to beattracted to their oppositely charged counterparts and potentially mayenter into both inter- and intramolecular ionic bonds. This non-covalentmechanism will tend to hold the linker into a conformation which allowsbonding between the oppositely charged groups. The addition of ancillarygroups which are charged, or alternatively, bear a latent charge whendeprotected, following addition to the linker, including deprotection ofa carboxyl, hydroxyl, thiol or amino group by a change in pH, oxidation,reduction or other mechanisms known to those skilled in the art whichresult in removal of the protecting group, is within the scope of thisinvention.

In view of the above, it is apparent that the appropriate selection of alinker group providing suitable orientation, restricted/unrestrictedrotation, the desired degree of hydrophobicity/hydrophilicity, etc. iswell within the skill of the art. Eliminating or reducing antigenicityof the multibinding compounds described herein is also within the scopeof this invention. In certain cases, the antigenicity of a multibindingcompound may be eliminated or reduced by use of groups such as, forexample, poly(ethylene glycol).

As explained above, the multibinding compounds described herein comprise2–10 ligands attached to a linker that links the ligands in such amanner that they are presented to the enzyme for multivalentinteractions with ligand binding sites thereon/therein. The linkerspatially constrains these interactions to occur within dimensionsdefined by the linker. This and other factors increases the biologicalactivity of the multibinding compound as compared to the same number ofligands made available in monobinding form.

The compounds of this invention are preferably represented by theempirical formula (L)_(p)(X)_(q) where L, X, p and q are as definedabove. This is intended to include the several ways in which the ligandscan be linked together in order to achieve the objective ofmultivalency, and a more detailed explanation is described below.

As noted previously, the linker may be considered as a framework towhich ligands are attached. Thus, it should be recognized that theligands can be attached at any suitable position on this framework, forexample, at the termini of a linear chain or at any intermediateposition.

The simplest and most preferred multibinding compound is a bivalentcompound which can be represented as L-X-L, where each L isindependently a ligand which may be the same or different and each X isindependently the linker. Examples of such bivalent compounds areprovided in FIG. 56 where each shaded circle represents a ligand. Atrivalent compound could also be represented in a linear fashion, i.e.,as a sequence of repeated units L-X-L-X-L, in which L is a ligand and isthe same or different at each occurrence, as can X. However, a trimercan also be a radial multibinding compound comprising three ligandsattached to a central core, and thus represented as (L)₃X, where thelinker X could include, for example, an aryl or cycloalkyl group.Illustrations of trivalent and tetravalent compounds of this inventionare found in FIGS. 57 and 58, respectively, where again, the shadedcircles represent ligands. Tetravalent compounds can be represented in alinear array, e.g.,L-X-L-X-L-X-Lin a branched array, e.g.,

(a branched construct analogous to the isomers of butane—n-butyl,iso-butyl, sec-butyl, and t-butyl) or in a tetrahedral array, e.g.,

where X and L are as defined herein. Alternatively, it could berepresented as an alkyl, aryl or cycloalkyl derivative as above withfour (4) ligands attached to the core linker.

The same considerations apply to higher multibinding compounds of thisinvention containing 5–10 ligands, as illustrated in FIG. 59. However,for multibinding agents attached to a central linker such as aryl orcycloalkyl, there is a self-evident constraint that there must besufficient attachment sites on the linker to accommodate the number ofligands present; for example, a benzene ring could not directlyaccommodate more than 6 ligands, whereas a multi-ring linker (e.g.,biphenyl) could accommodate a larger number of ligands.

Certain of the above described compounds may alternatively berepresented as cyclic chains of the form:

and variants thereof.

All of the above variations are intended to be within the scope of theinvention defined by the formula (L)_(p)(X)_(q).

With the foregoing in mind, a preferred linker may be represented by thefollowing formula:—X^(a)-Z-(Y^(a)-Z)_(m)-Y^(b)-Z-X^(a)—in which:

m is an integer of from 0 to 20;

X^(a) at each separate occurrence is selected from the group consistingof —O—, —S—, —NR—, —C(O)—, —C(O)O—, —C(O)NR—, —C(S), —C(S)O—, —C(S)NR—or a covalent bond where R is as defined below;

Z is at each separate occurrence is selected from the group consistingof alkylene, substituted alkylene, cycloalkylene, substitutedcylcoalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

Y^(a) and Y^(b) at each separate occurrence are selected from the groupconsisting of:

—S(O)_(n)—CR′R″——S(O)_(n)—NR′——S—S— or a covalent bond;in which:

n is 0, 1 or 2; and

R, R′ and R″ at each separate occurrence are selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic.

Additionally, the linker moiety can be optionally substituted at anyatom therein by one or more alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic group.

In one embodiment of this invention, the linker (i.e., X, X′ or X″) isselected those shown in Table II:

TABLE II Representative Linkers Linker—HN—(CH₂)₂—NH—C(O)—(CH₂)—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₂—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₄—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₅—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₆—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₇—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₈—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₉—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₁₀—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₁₁—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₁₂—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 1,2-phenyl—HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 1,3-phenyl—HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 1,4-phenyl—HN—(CH₂)₂—NH—C(O)—Z—O—Z—C(O)—NH—(CH₂)₂—NH— where Z is 1,4-phenyl—HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—(CH₂)₈—CH₃)—C(O)—NH—(CH₂)₂— NH——HN—(CH₂)₂—NH—C(O)—(CH₂)—O—(CH₂)—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is5-(n-octadecyloxy)-1,3-phenyl—HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—Z)—C(O)—NH—(CH₂)₂—NH— where Z is4-biphenyl —HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is5-(n-butyloxy)-1,3-phenyl—HN—(CH₂)₂—NH—C(O)—(CH₂)₈-trans-(CH═CH)—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—(CH₂)₁₂—CH₃)—C(O)—NH—(CH₂)₂— NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—Z)—C(O)—NH—(CH₂)₂—NH— where Z is4-(n-octyl)-phenyl —HN—(CH₂)—Z—O—(CH₂)₆—O—Z—(CH₂)—NH— where Z is1,4-phenyl—HN—(CH₂)₂—NH—C(O)—(CH₂)₂—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₂—C(O)—NH—(CH₂)₂—NH— —HN—(CH₂)₂—NH—C(O)—(CH₂)₂—CH(NH—C(O)—Ph)—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)—N+((CH₂)₉—CH₃)(CH₂—C(O)—NH—(CH₂)₂—NH₂)—(CH₂)—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)—N((CH₂)₉—CH₃)—(CH₂)—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—(CH₂)₂—NH—C(O)—(CH₂)₂—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₂—C(O)—NH—(CH₂)₂—C(O)—NH—(CH₂)₂—NH——HN—(CH₂)₂—NH—C(O)—Z—C(O)—NH—(CH₂)₂—NH— where Z is 5-hydroxy-1,3-phenyl

In another embodiment of this invention, the linker (i.e., X, X′ or X″)has the formula:

wherein

each R^(a) is independently selected from the group consisting of acovalent bond, alkylene, substituted alkylene and arylene;

each R^(b) is independently selected from the group consisting ofhydrogen, alkyl and substituted alkyl; and

n′ is an integer ranging from 1 to about 20.

In view of the above description of the linker, it is understood thatthe term “linker” when used in combination with the term “multibindingcompound” includes both a covalently contiguous single linker (e.g.,L-X-L) and multiple covalently non-contiguous linkers (L-X-L-X-L) withinthe multibinding compound.

Ligands

Any compound which binds to bacterial ribosomal RNA and/or to one ormore proteins involved in ribosomal protein synthesis in the bacterium,and preferably, which inhibits protein expression through such binding,can be used as a ligand to prepare the compounds described herein. Suchligands are well known to those of skill in the art.

Preferably, each ligand, L, in the multibinding compound of formula I isindependently selected from a compound of the formulas shown in FIG. 1.

The ligands shown in FIG. 1 (and the precursors/synthons thereof) arewell-known in the art and can be readily prepared using art-recognizedstarting materials, reagents and reaction conditions. By way ofillustration, the following patents and publications disclose compounds,intermediates and procedures useful in the preparation of ligands withthe formulas shown in FIG. 1 or related compounds suitable for use inthis invention:

Abonkhai, V. I. et al., In Vitro Activity of U-57930E, a New ClindamycinAnalog, Against Aerobic Gram-Positive Bacteria, Antimicrobial Agents andChemotherapy, 21(6):902–905 (1982).

Ballesta, J. P., et al., Peptidyltransferase inhibitors.Structure-Activity Relationship Analysis by Chemical Modification,Centro de Biologia Molecular, Chapter 44, pp 502–510, 199???. XAV

PCT WO 95/07271 by Barbachyn, p 1–33

Barden, T. C., et al., In Vitro Antibacterial Activity of9-(Glycylamido) tetracycline Derivatives, J. Med. Chem.,37(20):3205–3211 (1995).

Blackwood, R. K., et al., Some Transformations of Tetracycline at the4-Position, Can. J. Chem., 43:1382–1388 (1965).

Brickner, S. J., et al., Synthesis and Antibacterial Activity ofU-100592 and u-100766, Two Oxazolidinone Antibacterial Agents for thePotential Treatment of Multidrug-Resistant Gram-Positive BacterialInfections, J. Med. Chem., 39:673–679 (1986).

Cooperman, B. S. et al., Antibiotic Probes of Escherichia coli RibosomalPeptidyltransferase, Roche Institute of Molecular Biology, Chapter 42,pp 491–501, 199???. XAV

Cundliffe, Eric, Recognition Sites for Antibiotics within rRNA,Department of Biochemistry and Leicester Biocentre, LEI 7RH, UK, Chapter41, pp 479–510, 199???.XAV

Delaware, D. L., et al., Aminoglycoside Antibiotics (DihydrostreptomycinAnalogues), J. Antibiotics, XXXIX(2):251–258 (1986).

Herrinton, P. M., et al., Oxidation and Alkylation of SpectinomycinDerivatives; Synthesis of Trospectomycin from Spectinomycin, J. Org.Chem., 678–682 (1993).

Kirst, Ph.D., H. A., 2 Semi-synthetic derivatives of Erythromycin, Med.Chem., 30:57–88 (1993).

LemieuX, R. U., et al., The Chemistry of Streptomycin, J. Am. Chem.Soc., 52:337–385 (1947).

Mossa, J. S., et al., Streptomycin, Analytical Profiles of DrugSubstances, 16:507, 531–535,587 (1987).

Rosenbrook, Jr., W., et al., Spectinomycin Modification, ACS Symposium,Series 125, 1980.

Spahn, C. M. T., et al., Throwing a spanner in the works: antibioticsand the translation apparatus, Mol. Med., 74:423–430 (1996).

Thomas, R. C., et al., Synthesis of Spectinomycin Analogs, ACSSymposium, Series 125, 121–130 (1980).

Tohma, S., et al., Ashimycins A and B, new streptomycin Analogues, J.Antibiotic, XLII(8):1205–1212 (1989).

Tucker, J. A., et al., Piperazinyl Oxazolidinone Antibacterial AgentsContaining a pyridine, Diazene, or Triazene Heteroaromatic Ring, J. Med.Chem., 41:3727–3735 (1988).

White, D. R., et al., The Synthesis of Trospectomycin(6′-n-Propylspectinomycin, U-63,366F) From Spectinomycin, TetrahedronLetters, 30(12): 1469–1472 (1989).

Each of these patents and publications is incorporated herein byreference in its entirety to the same extent as if each individualpatent or publication was specifically and individually indicated to beincorporated by reference in its entirety.

Drugs such as chloramphenicol, erythromicin, clarithromycin,azithromycin, dirithromycin, flurithromycin clindamycin, lincomycin,quinopristin, dalfopristin, streptogramins, linezolid, U-100480,U-101603, U-94901, U-101244, pristinamycin, MJ-347-81F4A, HMR-3647,L-708299, A-184656, L-708365, L-701677, lexithromycin, RU-64004,CP-227182, CP-426027, TEA-0769, CP-279107, RU-56006, RU-6652, RU-59616,L-744434, L-744433, L-740893, L-709936, leucomycin, A-179796,eperezolid, and U-100480 inhibit ribosomal protein biosynthesis throughbinding to the 50S ribosomal subunit. They can be used to treatBacterial infections, generally, and, more specifically, Pneumocystiscarinii infections.

Drugs such as tetracycline, chlortetracycline, oxytetracycline,demeclocycline, methacycline, doxycycline, minocycline, CL-331002,glycylcyclines, CL-331928, CL-344667, CL-329998 and PAM-MINO inhibitribosomal protein biosynthesis through binding to the aminoacyl tRNAsite on the 30S ribosomal unit. They can be used to treat bacterialinfections, generally.

Drugs such as streptomycin, gentamicin, tobramycin, amikacin,netilimicin, kanamycin, neomycin, spectinomycin, dactimicin, paromomycinand trospectomycin inhibit ribosomal protein synthesis through bindingto the 30S ribosomal unit. They can be used to treat bacterialinfections, generally.

Drugs such as fusidic acid and purpuromycin inhibit protein synthesisthrough binding to soluble protein factors. They can be used to treatbacterial infections, generally.

Preparation of Multibinding Compounds

The multibinding compounds of this invention can be prepared fromreadily available starting materials using the following general methodsand procedures. It will be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Second Edition, Wiley, New York, 1991, andreferences cited therein.

Any compound which binds to bacterial ribosomal RNA and/or to one ormore proteins present in the bacterial ribosome, and, preferably, whichinhibits or otherwise adversely affects protein expression, can be usedas a ligand in this invention. As discussed in further detail below,numerous such compound are known in the art and any of these knowncompounds or derivatives thereof may be employed as ligands in thisinvention. Typically, a compound selected for use as a ligand will haveat least one functional group, such as an amino, hydroxyl, thiol orcarboxyl group and the like, which allows the compound to be readilycoupled to the linker. Compounds having such functionality are eitherknown in the art or can be prepared by routine modification of knowncompounds using conventional reagents and procedures. The patents andpublications set forth above provide numerous examples of suitablyfunctionalized macrolide antibiotics, aminoglycosides, lincosamides,oxazolidinones, streptogramins, tetracycline and other compounds whichbind to bacterial ribosomal RNA and/or to one or more proteins involvedin ribosomal protein synthesis in the bacterium, and intermediatesthereof, which may be used as ligands in this invention.

The ligands can be covalently attached to the linker through anyavailable position on the ligands, provided that when the ligands areattached to the linker, at least one of the ligands retains its abilityto bind to bacterial ribosomal RNA and/or to one or more proteinsinvolved in ribosomal protein synthesis in the bacterium. Certain sitesof attachment of the linker to the ligand are preferred based on knownstructure-activity relationships. Preferably, the linker is attached toa site on the ligand where structure-activity studies show that a widevariety of substituents are tolerated without loss of receptor activity.

It will be understood by those skilled in the art that the followingmethods may be used to prepare other multibinding compounds of thisinvention. Ligand precursors, for example, ligands containing a leavinggroup or a nucleophilic group, can be covalently linked to a linkerprecursor containing a nucleophilic group or a leaving group, usingconventional reagents and conditions.

As shown in Scheme A (FIG. 2), the reaction product of erythromyclamineand Fmoc protected glycinal followed by reduction of the resulting imineand deprotection (compound 103) can be reacted with a diacid underroutine amidation conditions to afford a ligand dimer.

As shown in Scheme B (FIG. 3), the amine in erythromyclamine can beprotected and a reactive hydroxy group reacted to form an imidazolidewhich can be reacted with a diamine to form a ligand dimer.

As shown in Scheme C (FIG. 4), an imidazolide can be reacted with anamino acid. The resulting compound has a carboxylic acid group which canbe coupled with the free amine group in (103) using routine conditions.

As shown in Scheme D (FIG. 5), an imidazolide can be reacted with twoequivalents of an amine to form a diurea compound.

As shown in Scheme E (FIG. 6), two equivalents of an amine-containingcompound can be reacted with a diacid such as adipic acid to form adi-amide.

As shown in Scheme F (FIG. 7), an imidazolide can be reacted with anamino acid to form a urea linkage, and the free carboxylic acid groupfrom the amino acid can be coupled with an amine to form a ligand dimer.

As shown in Scheme G (FIG. 8) and Scheme H (FIG. 9), two moleculescontaining amine groups can be reacted with a dibromide or a diacid toform the desired compound.

As shown in Scheme I (FIG. 10), two equivalents of a ligand containingan aldehyde group can be reacted with a linker containing two aminegroups, and the resulting imine reduced to form a diamine.

As shown in Scheme J (FIG. 11), a ligand containing an amine group canbe reacted with a linker containing an aldehyde group, and the resultingimine reduced to form a diamine.

As shown in Scheme K (FIG. 12), a ligand containing a secondary aminecan be reacted with an Fmoc protected glycine and the resulting aminedeprotected to form a primary amine derivative. Two equivalents of theprimary amine can be reacted with a diacid to form a ligand dimer.

As shown in Scheme L (FIG. 13), a ligand with an aromatic amine groupcan be reacted to form an imidazolide. Two equivalents of theimidazolide can be reactrd with a diamine to form a diurea compound.

As shown in Scheme M (FIG. 14), the imidazolide from scheme L can bereacted with an amino acid to form an amide linkage, and the freecarboxylic acid group reacted with an amine on a second ligand to createa dimer with mixed ligands.

As shown in Scheme N (FIG. 15), two equivalents of a ligand with analpha-beta unsaturated ketone moiety can be reacted with a dithiol toform the desired product after the thiols add across the double bond onthe ligands.

As shown in Scheme O (FIG. 16), two equivalents of a ligand with aprimary amine group can be reacted with a diacid under appropriateconditions to form a di-amide reaction product.

As shown in Scheme P (FIG. 17), one equivalent of a ligand with analpha-beta unsaturated ketone can be reacted with a linker including athiol group and a carboxylic acid group such that the thiol adds acrossthe double bond. The free carboxylic acid is then free to react with anamine group on a second ligand to form a dimer.

As shown in Scheme Q (FIG. 18), two equivalents of a ligand with anunsaturated ester linkage can also be reacted with a dithiol linkerunder conditions where the thiol groups add across the double bonds toform the desired product.

As shown in Scheme R (FIG. 19), one equivalent of a first ligandcontaining an alpha-beta unsaturated ketone and one equivalent of adithiol can be reacted such that the resulting product includes areactive thiol group, which can then be reacted with a second ligandcontaining an unsaturated ester moiety, to form a mixed dimer.

As shown in Scheme S (FIG. 20), one equivalent of a ligand containing anunsaturated ester moiety can be reacted with one equivalent of a linkerincluding a thiol and a carboxylic acid moiety to form an intermediatecontaining a free carboxylic acid group. This carboxylic acid group canthen be reacted with a free amine group on a second ligand to form amixed dimer.

As shown in Scheme T (FIG. 21), a ligand with more than one amine groupcan be reacted with a dihalide to form a mixture of dimers.

As shown in Scheme U (FIG. 22), a ligand with a primary amine group andone or more additional secondary amine groups can be selectively reactedwith an aldehyde group on a second ligand to form a dimer without theneed for an intermediate linker molecule.

As shown in Scheme V (FIG. 23), a ligand with a primary amine group andone or more additional secondary amine groups can be selectively reactedwith a linker molecule containing two acid groups to form a dimer.

As shown in Scheme W (FIG. 24), a ligand with an unprotected amine groupand one or more additional protected amine groups can be selectivelyreacted with a linker molecule with two bromo groups to form a dimer.

As shown in Scheme X (FIG. 24A), a ligand with an unprotected aminegroup and one or more additional protected amine groups can beselectively reacted with a linker molecule with a halide group and acarboxylic acid group to form an intermediate with a free carboxylicacid group, which can then be reacted with another ligand containing anamine group to form a mixed dimer.

As shown in Scheme Y (FIG. 25), one equivalent of a ligand with an aminegroup can be reacted with one equivalent of a linker with two acidgroups to form an intermediate with a free carboxylic acid group. Thisintermediate can be reacted with a second ligand which includes anunprotected amine group and one or more additional protected aminegroups to form a mixed dimer.

As shown in Scheme Z (FIG. 25A), a ligand with a secondary amine groupcan be reacted with a linker molecule containing a halide and acarboxylic acid group to form an intermediate with a free carboxylicacid group. The intermediate can then be reacted with a second ligandwith a free amine group to form a mixed dimer.

As shown in Scheme AA (FIG. 26), one equivalent of a ligand containingan imidazolide group (and in FIG. 26, a protected amine group) can bereacted with one equivalent of a linker molecule with two amine groupsto form an intermediate with a free amine group. This intermediate canbe reacted with a second ligand with an imidazolide group to form amixed dimer (in FIG. 26, the protected amine group was is thendeprotected).

As shown in Scheme BB (FIG. 27), one equivalent of a ligand with a freeprimary amine group can be reacted with one equivalent of a diacid toform an intermediate with a free carboxylic acid group. Thisintermediate can be reacted with a second ligand with a free amine groupto form a mixed dimer.

As shown in Scheme CC (FIG. 28), an intermediate ligand/dimer whichincludes a urethane group and a free carboxylic acid group can bereacted with a second ligand containing a free amine group to form mixeddimer.

As shown in Scheme DD (FIG. 29), one equivalent of a ligand with animidazolide or other activated carboxylic acid group can be reacted withone equivalent of a linker with two amine groups to form an intermediatewith a free amine group. This intermediate can be reacted with a secondligand with an imidazolide or other activated carboxylic acid group toform a mixed dimer.

As shown in Scheme EE (FIG. 30), one equivalent of a ligand with twofree amine groups can be reacted with a linker with a halide and acarboxylic acid group to form a mixture of intermediates with a freecarboxylic acid group. These intermediates can then be reacted with asecond ligand with a free amine group to form a mixture of liganddimers.

As shown in Scheme FF (FIG. 31), one equivalent of a ligand with animidazolide or other activated carboxylic acid group can be reacted withone equivalent of a linker with two amine groups to form an intermediatewith a free amine group, which can be reacted with a second ligand withan imidazolide or other activated carboxylic acid group to form a mixeddimer.

As shown in Scheme GG (FIG. 32), an intermediate formed by reacting afirst ligand containing an amine group with a diacid can be reacted witha second ligand with a free amine group to form a mixed dimer.

As shown in Scheme HH (FIG. 33), an intermediate formed by reacting afirst ligand containing an imidazolide group with a linker containing anamine group and a carboxylic acid can be reacted with a second ligandwith a free amine group to form a mixed dimer.

As shown in Scheme II (FIG. 34), an intermediate formed by reacting aligand containing an imidiazolide or other activated carboxylic acidgroup with a linker with two amine groups can be reacted with a secondligand with an imidiazolide or other activated carboxylic acid group toform a mixed dimer.

As shown in Scheme JJ (FIG. 35), a ligand with an amine group can bereacted with a linker with a halide and a carboxylic acid group to forman intermediate with a free halide group. This intermediate can bereacted with a second ligand with a free amine group to form a mixeddimer.

As shown in Scheme KK (FIG. 36), a ligand with an imidazolide or otheractive carboxylic acid group can be reacted with a linker with a primaryamine group and a halide group to form an intermediate with a halidegroup. The halide group can be reacted with an amine on a second ligandto form a mixed dimer.

As shown in Scheme LL (FIG. 37), a ligand with a secondary amine groupcan be reacted with a linker with a halide group and a carboxylic acidgroup to form an intermediate with a free carboxylic acid group. Thisintermediate can be reacted with a second ligand with a free amine groupto form a mixed dimer.

As shown in Scheme MM (FIG. 38), one equivalent of a ligand with asecondary amine group can be reacted with one equivalent of a dihalidelinker to form an intermediate which has a halide group. Thisintermediate can be reacted with a second ligand with two free aminogroups to form a mixture of mixed dimers.

As shown in Scheme NN (FIG. 39), one equivalent of a ligand with asecondary amine group can be reacted with a linker with a halide groupand a carboxylic acid group to form an intermediate which has a freecarboxylic acid group. This intermediate can be reacted with a secondligand with an amine group to form a mixed dimer.

As shown in Scheme OO (FIG. 40), an intermediate formed from a ligandcontaining an amine group and a linker with two carboxylic acid groupscan be reacted with a second ligand with an amine group to form a mixeddimer.

As shown in Scheme PP (FIG. 41), an intermediate formed by reacting aligand containing an unsaturated ketone group with a linker with a thioland a carboxylic acid group can be reacted with one equivalent of asecond ligand with an amine group to form a mixed dimer.

As shown in Scheme QQ (FIG. 42), an intermediate formed by reacting aligand containing an unsaturated ester group with a linker with a thioland a carboxylic acid group can be reacted with one equivalent of asecond ligand with an amine group to form a mixed dimer.

As shown in Scheme RR (FIG. 43), one equivalent of an intermediateformed by reacting a ligand with a secondary amine group with a linkerwith a halide group and a carboxylic acid group can be reacted with asecond ligand with an amine group to form a mixed dimer.

As shown in Scheme SS (FIG. 44), a ligand with a primary amine group canbe reacted with an intermediate formed by reacting a linker containing athiol group and a carboxylic acid group with a ligand with anunsaturated ester group to form a mixed dimer.

As shown in Scheme TT (FIG. 45), one equivalent of a ligand with anamine group can be reacted with one equivalent of a linker with twocarboxylic acid groups to form an intermediate with a free carboxylicacid group, which can be reacted with a second ligand with an aminegroup to form a mixed dimer.

As shown in Scheme UU (FIG. 46), an intermediate formed by reacting aligand containing an imidazolide group with a linker molecule containingan amine group and a carboxylic acid group can be reacted with a secondligand with a free amine group to form a mixed dimer.

As shown in Scheme VV (FIG. 47) and Scheme WW (FIG. 48), an intermediateformed by reacting a ligand containing an unsaturated ester group with alinker containing a thiol and a carboxylic acid group can be reactedwith a second ligand with an amine group to form a mixed dimer.

As shown in Scheme XX (FIG. 49), an intermediate formed by reacting aligand containing an unprotected amine group (and, as shown in thefigure, four protected amine groups) with a linker containing two halidegroups can be reacted with a ligand containing an amine group to form amixed dimer. The protected amine groups can then be deprotected.

As shown in Scheme YY (FIG. 50), a first ligand containing anunprotected amine group can be reacted with a dihalide linker to form anintermediate with a free halide group. This intermediate can be reactedwith a ligand with two unprotected amine groups to form a mixture ofmixed dimers. The protected amine groups can then be deprotected.

As shown in Scheme ZZ (FIG. 51), one equivalent of a ligand with anamine group can be reacted with one equivalent of a dihalide to form anintermediate with a free halide group. This intermediate can be reactedwith a second ligand with two free amine groups to form a mixture ofmixed dimers.

As shown in Scheme AAA (FIG. 52), two intermediates formed by reactingone equivalent of a ligand with two secondary amine groups with oneequivalent of a linker with a halide and a carboxylic acid group arereacted with a second ligand containing a primary amine group to form amixture of mixed dimers.

As shown in Scheme BBB (FIG. 53), one equivalent of a first ligandcontaining an amine group can be reacted with one equivalent of a linkermolecule with two isocyanate groups to form an intermediate with a urealinkage and a free isocyanate group. This intermediate can be reactedwith a second ligand with an amine group to form a mixed dimer with twourea linkages.

As shown in Scheme CCC (FIG. 54), a ligand with an amine group can becoupled with a linker with two carboxylic acid groups to form anintermediate with a free carboxylic acid group. This intermediate can bereacted with a second ligand with an amine group to form a mixed dimer.

As shown in Scheme DDD (FIG. 55), a ligand with an amine group can bereacted with a linker containing a halide group and a carboxylic acidgroup to form an intermediate with a free carboxylic acid group. Thisintermediate can be reacted with a second ligand with an amine group toform a mixed dimer.

In each of the above reaction schemes, a particular antibiotic orcombination thereof is listed. However, it is intended that theseschemes are applicable to other ligands with similar reactive groups.

Other methods are well known to those of skill in the art for couplingmolecules such as the ligands described herein with the linker moleculesdescribed herein. For example, two equivalents of ligand precursor witha halide, tosylate, or other leaving group, can be readily coupled to alinker precursor containing two nucleophilic groups, for example,phenoxide groups, to form a dimer. The leaving group employed in thisreaction may be any conventional leaving group including, by way ofexample, a halogen such as chloro, bromo or iodo, or a sulfonate groupsuch as tosyl, mesyl and the like. When the nucleophilic group is aphenol, any base which effectively deprotonates the phenolic hydroxylgroup may be used, including, by way of illustration, sodium carbonate,potassium carbonate, cesium carbonate, sodium hydride, sodium hydroxide,potassium hydroxide, sodium ethoxide, triethylamine,diisopropylethylamine and the like. Nucleophilic substitution reactionsare typically conducted in an inert diluent, such as tetrahydrofuran,N,N-dimethylformamide, N,N-dimethylacetamide, acetone, 2-butanone,1-methyl-2-pyrrolidinone and the like. After the reaction is complete,the dimer is typically isolated using conventional procedures, such asextraction, filtration, chromatography and the like.

By way of further illustration, dimers with a hydrophilic linker can beformed using a ligand precursor containing nucleophilic groups and apolyoxyethylene containing leaving groups, for example,poly(oxyethylene) dibromide (where the number of oxyethylene units istypically an integer from 1 to about 20). In this reaction, two molarequivalents of the ligand precursor are reacted with one molarequivalent of the poly(oxyethylene) dibromide in the presence of excesspotassium carbonate to afford a dimer. This reaction is typicallyconducted in N,N-dimethylformamide at a temperature ranging from about25° C. to about 100° C. for about 6 to about 48 hours.

Alternatively, the linker connecting the ligands may be prepared inseveral steps. Specifically, a ligand precursor can first be coupled toan “adapter”, i.e., a bifunctional group having a leaving group at oneend and another functional group at the other end which allows theadapter to be coupled to a intermediate linker group. In some cases, thefunctional group used to couple to the intermediate linker istemporarily masked with a protecting group (“PG”). Representativeexamples of adapters include, by way of illustration, tert-butylbromoacetate, 1-Fmoc-2-bromoethylamine, 1-trityl-2-bromoethanethiol,4-iodobenzyl bromide, propargyl bromide and the like. After the ligandprecursor is coupled to the adapter and the protecting group is removedfrom the adapter's functional group (if a protecting group is present)to form an intermediate, two molar equivalents of the intermediate arethen coupled with an intermediate linker to form a dimer.

Ligand precursors can be coupled with adapters which include bothleaving groups and protecting groups to form protected intermediates.The leaving group employed in this reaction may be any conventionalleaving group including, by way of example, a halogen such as chloro,bromo or iodo, or a sulfonate group such as tosyl, mesyl and the like.Similarly, any conventional protecting group may be employed including,by way of example, esters such as the methyl, tert-butyl, benzyl (“Bn”)and 9-fluorenylmethyl (“Fm”) esters.

Protected intermediates can then be deprotected using conventionalprocedures and reagents to afford deprotected intermediates. Forexample, tert-butyl esters are readily hydrolyzed with 95%trifluoroacetic acid in dichloromethane; methyl esters can be hydrolyzedwith lithium hydroxide in tetrahydrofuran/water; benzyl esters can beremoved by hydrogenolysis in the presence of a catalyst, such aspalladium on carbon; and 9-fluorenylmethyl esters are readily cleavedusing 20% piperidine in DMF. If desired, other well-known protectinggroups and deprotecting procedures may be employed in these reactions toform deprotected intermediates.

Similarly, ligand precursors having an adapter with an amine functionalgroup can be prepared. Ligand precursors can be coupled with adapterswhich include leaving groups and protected amine groups to affordprotected intermediates. The leaving group employed in this reaction maybe any conventional leaving group. Similarly, any conventional amineprotecting group may be employed including, by way of example, trityl,tert-butoxycarbonyl (“Boc”), benzyloxycarbonyl (“CBZ”) and9-fluorenylmethoxy-carbonyl (“Fmoc”). After coupling the adapter to theligand precursor, the resulting protected intermediate is deprotected toafford a ligand precursor including an amine group using conventionalprocedures and reagents. For example, a trityl group is readily removedusing hydrogen chloride in acetone; a Boc group is removed using 95%trifluoroacetic acid in dichloromethane; a CBZ group can be removed byhydrogenolysis in the presence of a catalyst, such as palladium oncarbon; and a 9-fluorenylmethoxycarbonyl group is readily cleaved using20% piperidine in DMF to afford the deblocked amine. Other well-knownamine protecting groups and deprotecting procedures may be employed inthese reactions to form amine-containing intermediates and relatedcompounds.

Ligand precursors having an adapter, for example, one including a freecarboxylic acid group or a free amine group, can be readily coupled tointermediate linkers having complementary functional groups to formmultibinding compounds as described herein. For example, when onecomponent includes a carboxylic acid group, and the other includes anamine group, the coupling reaction typically employs a conventionalpeptide coupling reagent and is conducted under conventional couplingreaction conditions, typically in the presence of a trialkylamine, suchas ethyldiisopropylamine. Suitable coupling reagents for use in thisreaction include, by way of example, carbodiimides, such asethyl-3-(3-dimethylamino)propylcarbodiimide (EDC),dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and thelike, and other well-known coupling reagents, such asN,N′-carbonyldiimidazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline(EEDQ), benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),O-(7-azabenzotriazol-1-yl)-N,N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and the like. Optionally, well-known couplingpromoters, such N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBT),1-hydroxy-7-azabenzotriazole (HOAT), N,N-dimethylaminopyridine (DMAP)and the like, may be employed in this reaction. Typically, this couplingreaction is conducted at a temperature ranging from about 0° C. to about60° C. for about 1 to about 72 hours in an inert diluent, such as THF,to afford the dimer.

The multibinding compounds described herein can also be prepared using awide variety of other synthetic reactions and reagents. For example,ligand precursors having aryliodide, carboxylic acid, amine and boronicacid functional groups can be prepared. Hydroxymethyl pyrrole can bereadily coupled under Mitsunobu reaction conditions to various phenolsto provide, after deprotection, functionalized intermediates. TheMitsunobu reaction is typically conducted by reacting hydroxymethylpyrrole and the appropriate phenol using diethyl azodicarboxylate (DEAD)and triphenylphosphine at ambient temperature for about 48 hours.Deprotection, if necessary, using conventional procedures and reagentsthen affords the functionalized intermediates.

The functionalized intermediates can be employed in the synthesis ofmultibinding compounds. For example, aryliodide intermediates can becoupled with bis-boronic acid linkers to provide dimers. Typically, thisreaction is conducted by contacting two molar equivalents of thearyliodide and one molar equivalent of the bis-boronic acid in thepresence of tetrakis(triphenylphosphine)palladium(0), sodium carbonateand water in refluxing toluene.

Aryliodide intermediates can also be coupled with acrylate intermediatesor alkyne intermediate to afford dimers. These reactions are typicallyconducted by contacting two molar equivalents of aryliodideintermediates with one molar equivalent of either acrylates or alkynesin the presence of dichlorobis(triphenylphosphine)palladium (II), copper(I) iodide and diisopropylethylamine in N,N-dimethylformamide to affordthe respective dimers.

As will be readily apparent to those of ordinary skill in the art, thesynthetic procedures described herein or those known in the art may bereadily modified to afford a wide variety of compounds within the scopeof this invention.

Combinatorial Libraries

The methods described herein lend themselves to combinatorial approachesfor identifying multimeric compounds which possess multibindingproperties.

Specifically, factors such as the proper juxtaposition of the individualligands of a multibinding compound with respect to the relevant array ofbinding sites on a target or targets is important in optimizing theinteraction of the multibinding compound with its target(s) and tomaximize the biological advantage through multivalency. One approach isto identify a library of candidate multibinding compounds withproperties spanning the multibinding parameters that are relevant for aparticular target. These parameters include: (1) the identity ofligand(s), (2) the orientation of ligands, (3) the valency of theconstruct, (4) linker length, (5) linker geometry, (6) linker physicalproperties, and (7) linker chemical functional groups.

Libraries of multimeric compounds potentially possessing multibindingproperties (i.e., candidate multibinding compounds) and comprising amultiplicity of such variables are prepared and these libraries are thenevaluated via conventional assays corresponding to the ligand selectedand the multibinding parameters desired. Considerations relevant to eachof these variables are set forth below:

Selection of Ligand(s)

A single ligand or set of ligands is (are) selected for incorporationinto the libraries of candidate multibinding compounds which library isdirected against a particular biological target or targets, i.e.,binding to bacterial ribosomal RNA and/or to one or more proteinsinvolved in ribosomal protein synthesis in the bacterium, preferably ina manner which inhibits or otherwise adversely affects proteinexpression. The only requirement for the ligands chosen is that they arecapable of interacting with the selected target(s). Thus, ligands may beknown drugs, modified forms of known drugs, substructures of known drugsor substrates of modified forms of known drugs (which are competent tointeract with the target), or other compounds. Ligands are preferablychosen based on known favorable properties that may be projected to becarried over to or amplified in multibinding forms. Favorable propertiesinclude demonstrated safety and efficacy in human patients, appropriatePK/ADME profiles, synthetic accessibility, and desirable physicalproperties such as solubility, logP, etc. However, it is crucial to notethat ligands which display an unfavorable property from among theprevious list may obtain a more favorable property through the processof multibinding compound formation; i.e., ligands should not necessarilybe excluded on such a basis. For example, a ligand that is notsufficiently potent at a particular target so as to be efficacious in ahuman patient may become highly potent and efficacious when presented inmultibinding form. A ligand that is potent and efficacious but not ofutility because of a non-mechanism-related toxic side effect may haveincreased therapeutic index (increased potency relative to toxicity) asa multibinding compound. Compounds that exhibit short in vivo half-livesmay have extended half-lives as multibinding compounds. Physicalproperties of ligands that limit their usefulness (e.g. poorbioavailability due to low solubility, hydrophobicity, hydrophilicity)may be rationally modulated in multibinding forms, providing compoundswith physical properties consistent with the desired utility.

Orientation: Selection of Ligand Attachment Points and Linking Chemistry

Several points are chosen on each ligand at which to attach the ligandto the linker. The selected points on the ligand/linker for attachmentare functionalized to contain complementary reactive functional groups.This permits probing the effects of presenting the ligands to theirtarget binding site(s) in multiple relative orientations, an importantmultibinding design parameter. The only requirement for choosingattachment points is that attaching to at least one of these points doesnot abrogate activity of the ligand. Such points for attachment can beidentified by structural information when available. For example,inspection of a co-crystal structure of a ligand bound to its targetallows one to identify one or more sites where linker attachment willnot preclude the ligand/target interaction. Alternatively, evaluation ofligand/target binding by nuclear magnetic resonance will permit theidentification of sites non-essential for ligand/target binding. See,for example, Fesik, et al., U.S. Pat. No. 5,891,643, the disclosure ofwhich is incorporated herein by reference in its entirety. When suchstructural information is not available, utilization ofstructure-activity relationships (SAR) for ligands will suggestpositions where substantial structural variations are and are notallowed. In the absence of both structural and SAR information, alibrary is merely selected with multiple points of attachment to allowpresentation of the ligand in multiple distinct orientations. Subsequentevaluation of this library will indicate what positions are suitable forattachment.

It is important to emphasize that positions of attachment that doabrogate the activity of the monomeric ligand may also be advantageouslyincluded in candidate multibinding compounds in the library providedthat such compounds bear at least one ligand attached in a manner whichdoes not abrogate intrinsic activity. This selection derives from, forexample, heterobivalent interactions within the context of a singletarget molecule. For example, consider a ligand bound to its target, andthen consider modifying this ligand by attaching to it a second copy ofthe same ligand with a linker which allows the second ligand to interactwith the same target at sites proximal to the first binding site, whichinclude elements of the target that are not part of the formal ligandbinding site and/or elements of the matrix surrounding the formalbinding site, such as the membrane. Here, the most favorable orientationfor interaction of the second ligand molecule may be achieved byattaching it to the linker at a position which abrogates activity of theligand at the first binding site. Another way to consider this is thatthe SAR of individual ligands within the context of a multibindingstructure is often different from the SAR of those same ligands inmomomeric form.

The foregoing discussion focused on bivalent interactions of dimericcompounds bearing two copies of the same ligand joined to a singlelinker through different attachment points, one of which may abrogatethe binding/activity of the monomeric ligand. It should also beunderstood that bivalent advantage may also be attained with heteromericconstructs bearing two different ligands that bind to common ordifferent targets.

Once the ligand attachment points have been chosen, one identifies thetypes of chemical linkages that are possible at those points. The mostpreferred types of chemical linkages are those that are compatible withthe overall structure of the ligand (or protected forms of the ligand)readily and generally formed, stable and intrinsically innocuous undertypical chemical and physiological conditions, and compatible with alarge number of available linkers. Amide bonds, ethers, amines,carbamates, ureas, and sulfonamides are but a few examples of preferredlinkages.

Linker Selection

In the library of linkers employed to generate the library of candidatemultibinding compounds, the selection of linkers employed in thislibrary of linkers takes into consideration the following factors:

Valency: In most instances the library of linkers is initiated withdivalent linkers. The choice of ligands and proper juxtaposition of twoligands relative to their binding sites permits such molecules toexhibit target binding affinities and specificities more than sufficientto confer biological advantage. Furthermore, divalent linkers orconstructs are also typically of modest size such that they retain thedesirable biodistribution properties of small molecules.

Linker Length: Linkers are chosen in a range of lengths to allow thespanning of a range of inter-ligand distances that encompass thedistance preferable for a given divalent interaction. In some instancesthe preferred distance can be estimated rather precisely fromhigh-resolution structural information of targets. In other instanceswhere high-resolution structural information is not available, one canmake use of simple models to estimate the maximum distance betweenbinding sites either on adjacent receptors or at different locations onthe same receptor. In situations where two binding sites are present onthe same target (or target subunit for multisubunit targets), preferredlinker distances are 2–20 Å, with more preferred linker distances of3–12 Å. In situations where two binding sites reside on separate targetsites, preferred linker distances are 20–100 Å, with more preferreddistances of 30–70 Å.

Linker Geometry and Rigidity: The combination of ligand attachment site,linker length, linker geometry, and linker rigidity determine thepossible ways in which the ligands of candidate multibinding compoundsmay be displayed in three dimensions and thereby presented to theirbinding sites. Linker geometry and rigidity are nominally determined bychemical composition and bonding pattern, which may be controlled andare systematically varied as another spanning function in a multibindingarray. For example, linker geometry is varied by attaching two ligandsto the ortho, meta, and para positions of a benzene ring, or in cis- ortrans-arrangements at the 1,1-vs. 1,2-vs. 1,3-vs. 1,4-positions around acyclohexane core or in cis- or trans-arrangements at a point of ethyleneunsaturation. Linker rigidity is varied by controlling the number andrelative energies of different conformational states possible for thelinker. For example, a divalent compound bearing two ligands joined by1,8-octyl linker has many more degrees of freedom, and is therefore lessrigid than a compound in which the two ligands are attached to the 4,4′positions of a biphenyl linker.

Linker Physical Properties: The physical properties of linkers arenominally determined by the chemical constitution and bonding patternsof the linker, and linker physical properties impact the overallphysical properties of the candidate multibinding compounds in whichthey are included. A range of linker compositions is typically selectedto provide a range of physical properties (hydrophobicity,hydrophilicity, amphiphilicity, polarization, acidity, and basicity) inthe candidate multibinding compounds. The particular choice of linkerphysical properties is made within the context of the physicalproperties of the ligands they join and preferably the goal is togenerate molecules with favorable PK/ADME properties. For example,linkers can be selected to avoid those that are too hydrophilic or toohydrophobic to be readily absorbed and/or distributed in vivo.

Linker Chemical Functional Groups: Linker chemical functional groups areselected to be compatible with the chemistry chosen to connect linkersto the ligands and to impart the range of physical properties sufficientto span initial examination of this parameter.

Combinatorial Synthesis

Having chosen a set of n ligands (n being determined by the sum of thenumber of different attachment points for each ligand chosen) and mlinkers by the process outlined above, a library of (n!)m candidatedivalent multibinding compounds is prepared which spans the relevantmultibinding design parameters for a particular target. For example, anarray generated from two ligands, one which has two attachment points(A1, A2) and one which has three attachment points (B1, B2, B3) joinedin all possible combinations provide for at least 15 possiblecombinations of multibinding compounds:

A1-A1 A1-A2 A1-B1 A1-B2 A1-B3 A2-A2 A2-B1 A2- B2 A2-B3 B1-B1 B1-B2 B1-B3B2-B2 B2-B3 B3-B3When each of these combinations is joined by 10 different linkers, alibrary of 150 candidate multibinding compounds results.

Given the combinatorial nature of the library, common chemistries arepreferably used to join the reactive functionalies on the ligands withcomplementary reactive functionalities on the linkers. The librarytherefore lends itself to efficient parallel synthetic methods. Thecombinatorial library can employ solid phase chemistries well known inthe art wherein the ligand and/or linker is attached to a solid support.Alternatively and preferably, the combinatorial libary is prepared inthe solution phase. After synthesis, candidate multibinding compoundsare optionally purified before assaying for activity by, for example,chromatographic methods (e.g., HPLC).

Analysis of the Library

Various methods are used to characterize the properties and activitiesof the candidate multibinding compounds in the library to determinewhich compounds possess multibinding properties. Physical constants suchas solubility under various solvent conditions and logD/clogD values canbe determined. A combination of NMR spectroscopy and computationalmethods is used to determine low-energy conformations of the candidatemultibinding compounds in fluid media. The ability of the members of thelibrary to bind to the desired target and other targets is determined byvarious standard methods, which include radioligand displacement assaysfor receptor and ion channel targets, and kinetic inhibition analysisfor many enzyme targets. In vitro efficacy, such as for receptoragonists and antagonists, ion channel blockers, and antimicrobialactivity, can also be determined. Pharmacological data, including oralabsorption, everted gut penetration, other pharmacokinetics parametersand efficacy data can be determined in appropriate models. In this way,key structure-activity relationships are obtained for multibindingdesign parameters which are then used to direct future work.

The members of the library which exhibit multibinding properties, asdefined herein, can be readily determined by conventional methods. Firstthose members which exhibit multibinding properties are identified byconventional methods as described above including conventional assays(both in vitro and in vivo).

Second, ascertaining the structure of those compounds which exhibitmultibinding properties can be accomplished via art recognizedprocedures. For example, each member of the library can be encrypted ortagged with appropriate information allowing determination of thestructure of relevant members at a later time. See, for example, Dower,et al., International Patent Application Publication No. WO 93/06121;Brenner, et al., Proc. Natl. Acad. Sci., USA, 89:5181 (1992); Gallop, etal., U.S. Pat. No. 5,846,839; each of which are incorporated herein byreference in its entirety. Alternatively, the structure of relevantmultivalent compounds can also be determined from soluble and untaggedlibaries of candidate multivalent compounds by methods known in the artsuch as those described by Hindsgaul, et al., Canadian PatentApplication No. 2,240,325 which was published on Jul. 11, 1998. Suchmethods couple frontal affinity chromatography with mass spectroscopy todetermine both the structure and relative binding affinities ofcandidate multibinding compounds to receptors.

The process set forth above for dimeric candidate multibinding compoundscan, of course, be extended to trimeric candidate compounds and higheranalogs thereof.

Follow-up Synthesis and Analysis of Additional Libraries

Based on the information obtained through analysis of the initiallibrary, an optional component of the process is to ascertain one ormore promising multibinding “lead” compounds as defined by particularrelative ligand orientations, linker lengths, linker geometries, etc.Additional libraries can then be generated around these leads to providefor further information regarding structure to activity relationships.These arrays typically bear more focused variations in linker structurein an effort to further optimize target affinity and/or activity at thetarget (antagonism, partial agonism, etc.), and/or alter physicalproperties. By iterative redesign/analysis using the novel principles ofmultibinding design along with classical medicinal chemistry,biochemistry, and pharmacology approaches, one is able to prepare andidentify optimal multibinding compounds that exhibit biologicaladvantages towards their targets and as therapeutic agents.

To further elaborate upon this procedure, suitable divalent linkersinclude, by way of example only, those derived from dicarboxylic acids,disulfonylhalides, dialdehydes, diketones, dihalides, diisocyanates,diamines, diols, mixtures of carboxylic acids, sulfonylhalides,aldehydes, ketones, halides, isocyanates, amines and diols. In eachcase, the carboxylic acid, sulfonylhalide, aldehyde, ketone, halide,isocyanate, amine and diol functional group is reacted with acomplementary functionality on the ligand to form a covalent linkage.Such complementary functionality is well known in the art as illustratedin the following table:

Representative Complementary Binding Chemisties First Reactive GroupSecond Reactive Group Linkage hydroxyl isocyanate urethane amine epoxideβ-hydroxyamine sulfonyl halide amine sulfonamide carboxyl acid amineamide hydroxyl alkyl/aryl halide ether aldehyde amine(+reducing agent)amine ketone amine(+reducing agent) amine amine isocyanate urea

Exemplary linkers include the following linkers identified as X-1through X-418 as set forth below:

Pharmaceutical Formulations

When employed as pharmaceuticals, the compounds of this invention areusually administered in the form of pharmaceutical compositions. Thesecompounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. These compounds are effective as both injectable and oralcompositions. Such compositions are prepared in a manner well known inthe pharmaceutical art and comprise at least one active compound.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds described hereinassociated with pharmaceutically acceptable carriers. In making thecompositions of this invention, the active ingredient is usually mixedwith an excipient, diluted by an excipient or enclosed within such acarrier which can be in the form of a capsule, sachet, paper or othercontainer. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g., about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 0.001 to about 1 g, more usually about 1 toabout 30 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient. Preferably, the compound of formula I above is employed at nomore than about 20 weight percent of the pharmaceutical composition,more preferably no more than about 15 weight percent, with the balancebeing pharmaceutically inert carrier(s).

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It, willbe understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

The following formulation examples illustrate representativepharmaceutical compositions of the present invention.

FORMULATION EXAMPLE 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

FORMULATION EXAMPLE 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

FORMULATION EXAMPLE 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Active Ingredient 5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling appliance.

FORMULATION EXAMPLE 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows:

Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mg Starch 45.0 mgMicrocrystalline cellulose 35.0 mg Polyvinylpyrrolidone  4.0 mg (as 10%solution in sterile water) Sodium carboxymethyl starch  4.5 mg Magnesiumstearate  0.5 mg Talc  1.0 mg Total  120 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

FORMULATION EXAMPLE 5

Capsules, each containing 40 mg of medicament are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient  40.0 mg Starch 109.0mg Magnesium stearate  1.0 mg Total 150.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 150 mg quantities.

FORMULATION EXAMPLE 6

Suppositories, each containing 25 mg of active ingredient are made asfollows:

Ingredient Amount Active Ingredient   25 mg Saturated fatty acidglycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

FORMULATION EXAMPLE 7

Suspensions, each containing 50 mg of medicament per 5.0 mL dose aremade as follows:

Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodiumcarboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mgSucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purifiedwater to 5.0 mL

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

FORMULATION EXAMPLE 8

A formulation may be prepared as follows:

Quantity Ingredient (mg/capsule) Active Ingredient  15.0 mg Starch 407.0mg Magnesium stearate  3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 425.0 mg quantities.

FORMULATION EXAMPLE 9

A formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 5.0 mg Corn Oil 1.0 mL

FORMULATION EXAMPLE 10

A topical formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 1–10 g Emulsifying Wax 30 g LiquidParaffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference in its entirety. Such patches may be constructed forcontinuous, pulsatile, or on demand delivery of pharmaceutical agents.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985).

Utility

The multibinding compounds of this invention are macrolide antibiotics,aminoglycosides, lincosamides, oxazolidinones, streptogramins,tetracycline or other compounds which are known to bind bacterialribosomal RNA and/or one or more proteins involved in ribosomal proteinsynthesis in the bacterium. Accordingly, the multibinding compounds andpharmaceutical compositions of this invention are useful in thetreatment and prevention of bacterial infections.

Examples of bacterial infections which can be treated using thecompounds described herein include gram positive, gram negative andanaerobic bacterial infections.

When used in treating or ameliorating such conditions, the compounds ofthis invention are typically delivered to a patient in need of suchtreatment by a pharmaceutical composition comprising a pharmaceuticallyacceptable diluent and an effective amount of at least one compound ofthis invention. The amount of compound administered to the patient willvary depending upon what compound and/or composition is beingadministered, the purpose of the administration, such as prophylaxis ortherapy, the state of the patient, the manner of administration, and thelike.

In therapeutic applications, compositions are administered to a patientalready suffering from a bacterial infection. Amounts effective for thisuse will depend on the judgment of the attending clinician dependingupon factors such as the degree or severity of the disorder in thepatient, the age, weight and general condition of the patient, and thelike. The pharmaceutical compositions of this invention may contain morethan one compound of the present invention.

As noted above, the compounds administered to a patient are in the formof pharmaceutical compositions described above which can be administeredby a variety of routes including oral, rectal, transdermal,subcutaneous, intravenous, intramuscular, etc. These compounds areeffective as both injectable and oral deliverable pharmaceuticalcompositions. Such compositions are prepared in a manner well known inthe pharmaceutical art and comprise at least one active compound.

The multibinding compounds of this invention can also be administered inthe form of pro-drugs, i.e., as derivatives which are converted into abiologically active compound in vivo. Such pro-drugs will typicallyinclude compounds in which, for example, a carboxylic acid group, ahydroxyl group or a thiol group is converted to a biologically liablegroup, such as an ester, lactone or thioester group which will hydrolyzein vivo to reinstate the respective group.

The compounds can be assayed to identify which of the multimeric ligandcompounds possess multibinding properties. First, one identifies aligand or mixture of ligands which each contain at least one reactivefunctionality and a library of linkers which each include at least twofunctional groups having complementary reactivity to at least one of thereactive functional groups of the ligand. Next one prepares a multimericligand compound library by combining at least two stoichiometricequivalents of the ligand or mixture of ligands with the library oflinkers under conditions wherein the complementary functional groupsreact to form a covalent linkage between the linker and at least two ofthe ligands. The multimeric ligand compounds produced in the library canbe assayed to identify multimeric ligand compounds which possessmultibinding properties. The method can also be performed using alibrary of ligands and a linker or mixture of linkers.

The preparation of the multimeric ligand compound library can beachieved by either the sequential or concurrent combination of the twoor more stoichiometric equivalents of the ligands with the linkers. Themultimeric ligand compounds can be dimeric, for example, homodimeric orheteromeric. A heteromeric ligand compound library can be prepared bysequentially adding a first and second ligand.

Each member of the multimeric ligand compound library can be isolatedfrom the library, for example, by preparative liquid chromatography massspectrometry (LCMS). The linker or linkers can be flexible linkers,rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers ofdifferent geometry, acidic linkers, basic linkers, linkers of differentpolarizability and/or polarization or amphiphilic linkers. The linkerscan include linkers of different chain lengths and/or which havedifferent complementary reactive groups. In one embodiment, the linkersare selected to have different linker lengths ranging from about 2 to100 Å. The ligand or mixture of ligands can have reactive functionalityat different sites on the ligands. The reactive functionality can be,for example, carboxylic acids, carboxylic acid halides, carboxyl esters,amines, halides, pseudohalides, isocyanates, vinyl unsaturation,ketones, aldehydes, thiols, alcohols, anhydrides, boronates, andprecursors thereof, as long as the reactive functionality on the ligandis complementary to at least one of the reactive groups on the linker sothat a covalent linkage can be formed between the linker and the ligand.

A library of multimeric ligand compounds can thus be formed whichpossesses multivalent properties.

Multimeric ligand compounds possessing multibinding properties can beidentified in an iterative method by preparing a first collection oriteration of multimeric compounds by contacting at least twostoichiometric equivalents of the ligand or mixture of ligands whichtarget bacterial ribosomal RNA and/or one or more proteins involved inribosomal protein synthesis in the bacterium with a linker or mixture oflinkers, where the ligand or mixture of ligands includes at least onereactive functionality and the linker or mixture of linkers includes atleast two functional groups having complementary reactivity to at leastone of the reactive functional groups of the ligand. The ligand(s) andlinker(s) are reacted under conditions which form a covalent linkagebetween the linker and at least two of the ligands. The first collectionor iteration of multimeric compounds can be assayed to assess which ifany of the compounds possess multibinding properties. The process can berepeated until at least one multimeric compound is found to possessmultibinding properties. By evaluating the particular molecularconstraints which are imparted or are consistent with impartingmultibinding properties to the multimeric compound or compounds in thefirst iteration, a second collection or iteration of multimericcompounds which elaborates upon the particular molecular constraints canbe assayed, and the steps optionally repeated to further elaborate uponsaid molecular constraints. For example, the steps can be repeated frombetween 2 and 50 times, more preferably, between 5 and 50 times.

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention. Unless otherwise stated, alltemperatures are in degrees Celsius.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning.

Å = Angstroms cm = centimeter DCC = dicyclohexyl carbodiimide DMF =N,N-dimethylformamide DMSO = dimethylsulfoxide EDTA =ethylenediaminetetraacetic acid g = gram HPLC = high performance liquidchromatography MEM = minimal essential medium mg = milligram MIC =minimum inhibitory concentration min = minute mL = milliliter mm =millimeter mmol = millimol N = normal THF = tetrahydrofuran μL =microliters μm = microns

ANALYTICAL EXAMPLES Example 1

Binding Test

Methods for performing affinity labeling studies, for example, withchloramphenicol, are known to those of skill in the art. See, forexample, Cooperman, “Functional sites on the E. coli ribosome as definedby affinity labeling,” p. 531–554 (1980), Chambliss et al., inRibosomes—Structure, Function and Genetics, University Park Press,Baltimore; Cooperman, Affinity labeling of ribosomes, Methods Enzymol.,164:341–361 (1988); and Jaynes et al., Biochemistry, 17:561–569 (1978).Such methods can be used to determine the binding of the compounds ofthe present invention to various positions on ribosomal RNA and/or toproteins involved in bacterial protein synthesis.

BIOLOGICAL EXAMPLES Example 1

Determination of Antibacterial Activity

In Vitro Determination of Antibacterial Activity

Bacteria are obtained and phenotyped based on their sensitivity todifferent antibiotics including those that interfere with ribosomalprotein synthesis. Minimal inhibitory concentrations (MICs) are measuredin a microdilution broth procedure under NCCLS guidelines. The compoundsare serially diluted into Mueller-Hinton broth in 96-well microtiterplates. Overnight cultures of bacterial strains are diluted based onabsorbance at 600 nm so that the final concentration in each well was5×10⁵ cfu/ml. Plates are returned to a 35° C. incubator. The followingday (or 24 hours in the case of Enterococci strains), MICs aredetermined by visual inspection of the plates.

Bacterial strains which may be tested in this model include, but are notlimited to, those described herein. Growth conditions may be modified asnecessary for each particular strain. Growing conditions and growthmedia for the strains described herein are known in the art.

Determination of Kill Time

Experiments to determine the time required to kill the bacteria areconducted. These experiments are conducted with both staphylococcus andenterococcus strains.

Briefly, several colonies are selected from an agar plate and grown at35° C. under constant agitation until a turbidity of approximately1.5×10⁸ CFU/ml is achieved. The sample is diluted to about 6×10⁶ CFU/mland incubated at 35° C. under constant agitation. At various times,aliquots are removed and five ten-fold serial dilutions are performed.The pour plate method is used to determine the number of colony formingunits (CFUs).

In Vivo Determination of Antibacterial Activity

Acute Tolerability Studies in Mice

In these studies, the compounds to be evaluated are administered eitherintravenously or subcutaneously and observed for 5–15 minutes. If thereare no adverse effects, the dose is increased in a second group of mice.This dose incrementation continues until mortality occurs, or the doseis maximized. Generally, dosing begins at 20 mg/kg and increases by 20mg/kg each time until the maximum tolerated dose (MTD) is achieved.

Bioavailability Studies in Mice

Mice are administered the compound to be evaluated either intravenouslyor subcutaneously at a therapeutic dose (in general, approximately 50mg/kg). Groups of animals are placed in metabolic cages so that urineand feces may be collected for analysis. Groups of animals (n=3) aresacrificed at various times (10 min, 1 hour and 4 hours). Blood iscollected by cardiac puncture and the following organs are harvested:lung, liver, heart, brain, kidney, and spleen. Tissues were weighed andprepared for HPLC analysis. HPLC analysis on the tissue homogenates andfluids is used to determine the concentration of the compound. Metabolicproducts resulting from changes to the compound are also determined.

Mouse Septecemia Model

In this model, an appropriately virulent strain of bacteria (mostcommonly S. aureus, or E. faecalis or E. faecium) is administeredintraperitoneally to mice (N=5 to 10 mice per group). The bacteria wascombined with hog gastric mucin to enhance virulence. The dose ofbacteria (normally 10⁵–10⁷) is that which is sufficient to inducemortality in all of the mice over a three day period. One hour after thebacteria is administered, the compound to be evaluated is administeredin a single dose, either IV or subcutaneously. Each dose is administeredto groups of 5 to 10 mice, at doses that typically range from a maximumof about 20 mg/kg to a minimum of less than 1 mg/kg. A positive control(normally β-lactam with β-lactam sensitive strains) is administered ineach experiment. The dose at which approximately 50% of the animals aresaved is calculated from the results.

Neutropenic Thigh Model

In this model, antibacterial activity of the compound to be evaluated isevaluated against an appropriately virulent strain of bacteria (mostcommonly S. aureus). Mice are initially rendered neutropenic byadministration of cyclophosphamide at 200 mg/kg on day 0 and day 2. Onday 4, they are infected in the left anterior thigh by an IM injectionof a single dose of bacteria. The mice are administered the compound onehour after the administration of bacteria. At various later times(normally 1, 2.5, 4 and 24 hours) the mice are sacrificed (3 per timepoint). The thigh is excised, homogenized and the number of CFUs (colonyforming units) is determined by plating. Blood is also plated todetermine the CFUs in the blood.

Pharmacokinetic Studies

The rate at which the compound to be evaluated is removed from the bloodcan be determined in either rats or mice. In rats, the test animals arecannulated in the jugular vein. A compound is administered via tail veininjection, and at various time points (normally 5, 15, 30, 60 minutesand 2, 4, 6 and 24 hours) blood is withdrawn from the cannula. In mice,a compound is also administered via tail vein injection, and at varioustime points. Blood is normally obtained by cardiac puncture. Theconcentration of the remaining compound is determined by HPLC.

PREPARATIVE EXAMPLES Example 1

Preparation of (113), a Compound of Formula I Via Scheme A.

Erythromyclamine (100) (10 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (20 mmol) and Fmoc glycinal (101) (10 mmol)(prepared as described by Salvi et al. Tetrahedron Lett. 1994, 35,1181–1184). After 2 hours the reaction mixture is cooled in an ice waterbath and treated further with sodium cyanoborohydride (4.0 mmol) andtrifluoroacetic acid (30 mmol). After 2 additional hours the crudeproduct is concentrated under reduced pressure and fractionated byreverse-phase HPLC to afford the desired product (102).

The above product (102) is then dissolved in anhydrous dimethylformamide(10 mL), stirred at room temperature and treated with excess piperidine(1.0 mL). After one hour the crude products are concentrated underreduced pressure and fractionated by reverse-phase HPLC to afford thedesired product (103).

Succinic acid (104) (2.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated diacidis treated with compound (103) (4.0 mmol) and the coupling reactionmixture is stirred overnight at room temperature. Volatiles are removedunder vacuum and the crude is fractionated by reverse-phase HPLC toafford the title product after (lyopholization of the appropriatefractions.

The chemistry is detailed below in the following reaction scheme:

Example 2

Preparation of (108), a Compound of Formula II Via Scheme B.

Erythromyclamine (100) (34.0 mmol) is dissolved in THF under a nitrogenatmosphere. Di-tert-butyl dicarbonate (Boc₂O) (68.0 mmol) dissolved indichloromethane is added dropwise to the stirred solution. The course ofthe reaction is followed by TLC and stirring is continued at roomtemperature until the reaction is judged complete. The reaction mixtureis evaporated giving a precipitate that is collected by filtration. Theprecipitate is rinsed with ether to afford the desired product, which istreated with acetic anhydride in dichloromethane and purified by silicagel chromatography. The product (10 mmol) is then dissolved in toluene,stirred in an ice/water bath, and treated sequentially with K₂CO₃ (100mmol) and carbonyldiimidazole (10 mmol). The ice bath is removed and thereaction mixture is allowed to warm to room temperature. The imidazolide(105) thus produced is used without further manipulation in the couplingreactions described below.

1,4-Diaminobutane (106) (2.0 mmol) is dissolved in toluene/DMF, stirredat room temperature, and treated sequentially with diisopropylethylamine (4.0 mmol) and imidazolide (105) (4.0 mmol) prepared above. After2 hours, volatiles are removed under vacuum and the crude product ispurified by silica gel chromatography to afford the desired product(107).

Compound (107) (2.0 mmol) is dissolved in THF. Trimethylsilyl triflate(20 mmol) and lutidine (30 mmol) are added and the reaction is followedby TLC. When judged complete, the mixture is treated withtetrabutylammonium fluoride (30 mmol) and the reaction is followed byTLC. When judged complete, the mixture is diluted two-fold with methanoland heated at refux for 1 hour. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 3

Preparation of (112), a Compound of Formula III Via Scheme C.

A solution of (105) (6 mmol), prepared as described in Example 2, and6-aminocaproic acid (109) (6 mmol) in DMF. The course of the reaction isfollowed by thin layer chromatography. When reaction has occurred, thereaction mixture is concentrated under reduced pressure to give thecrude product. The desired compound (110) is obtained by purification ofthe crude product by use of HPLC.

Compound (110) (4.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with compound (103) (4.0mmol), hydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0mmol) and PyBOP (4.0 mmol) and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the desiredproduct (111) after lyopholization of the appropriate fractions.

Compound (111) (2.0 mmol) is dissolved in THF. Trimethylsilyl triflate(20 mmol) and lutidine (30 mmol) are added and the reaction is followedby TLC. When judged complete, the mixture is treated withtetrabutylammonium fluoride (30 mmol) and the reaction followed by TLC.When judged complete, the mixture is diluted two-fold with methanol andheated at reflux for 1 hour. Volatiles are removed under vacuum and thecrude is fractionated by reverse-phase HPLC to afford the title productafter lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 4

Preparation of (203), a Compound of Formula IV Via Scheme D.

Compound (201) (1.0 mmol) is dissolved in toluene, stirred in anice/water bath, and treated sequentially with K₂CO₃ (10 mmol) andcarbonyldiimidazole (1.0 mmol). The ice bath is removed and the reactionmixture is allowed to warm to room temperature. The imidazolide (202)thus produced is used without further manipulation in the couplingreactions described below.

A solution of (202) (1.0 mmol) in toluene/DMF with 1,4-diaminobutane(106) (0.5 mmol) is heated as necessary in a sealed vessel and thereaction followed by TLC. When judged complete, the mixture ispartitioned between ethyl acetate and water and the organic phase washedwith water, dried over sodium sulfate and the solvent removed in vacuo.The residue is purified by silica gel chromatography to afford the titleproduct.

Compound (201) is reported in J. Med. Chem. 1998, 41, 3727–3735.

The chemistry is detailed below in the following reaction scheme:

Example 5

Preparation of (206), a Compound of Formula V Via Scheme E.

Adipic acid (205) (2.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with compound (204) (4.0mmol), hydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0mmol) and PyBOP (4.0 mmol) and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe product is purified by silica gel chromatography to afford the titleproduct.

Compound (204) is reported in J. Med. Chem. 1996, 49, 637–679.

The chemistry is detailed below in the following reaction schenme:

Example 6

Preparation of (208), a Compound of Formula VI Via Scheme F.

A solution of (202) (6 mmol), prepared as described in Example 4, and6-aminocaproic acid (109) (6 mmol) in toluene/DMF is prepared underargon in a flask equipped with magnetic stirrer and drying tube. Thecourse of the reaction is followed by thin layer chromatography. Whenreaction has occurred, the reaction solution is diluted with ethylacetate and washed with aqueous HCl and then water. The organic layer isdried (Na₂SO₄), filtered and concentrated under reduced pressure to givethe crude product. The desired compound (207) is obtained bypurification of the crude product by use of HPLC.

Compound (207) (4.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with compound (204) (4.0mmol), hydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0mmol) and PyBOP (4.0 mmol) and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

Compound (204) is reported in J. Med. Chem. 1996, 49, 673–679.

The chemistry is detailed below in the following reaction scheme:

Example 7

Preparation of (303), a Compound of Formula VII Via Scheme G.

A solution of 20 mmols of compound (301) in DMF with 10 mmols of1,6-dibromohexane (302) and 40 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When reaction has occurred,the reaction mixture is concentrated under reduced pressure to give thecrude product, which is fractionated by reverse-phase HPLC to afford thetitle product after lyopholization of the appropriate fractions.

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother., 21:902–905 (1982).

The chemistry is detailed below in the following reaction scheme:

Example 8

Preparation of (403), a Compound of Formula VIII Via Scheme H.

A solution of 20 mmols of Streptomycin (401) in DMF with 10 mmols of1,4-dibromobutane (402) and 40 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When reaction has occurred,the reaction mixture is concentrated under reduced pressure to give thecrude product, which is fractionated by reverse-phase HPLC to afford thetitle product after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 9

Preparation of (404), a Compound of Formula VIII Via Scheme H.

Adipic acid (205) (2.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated diacidis treated with Streptomycin (401) (4.0 mmol) and the coupling reactionmixture is stirred overnight at room temperature. Volatiles are removedunder vacuum and the crude is fractionated by reverse-phase HPLC toafford the title product after lyopholization of the appropriatefractions.

The chemistry is detailed below in the following reaction scheme:

Example 10

Preparation of (406), a Compound of Formula IX Via Scheme I.

1,4-Diaminobutane (106) (10 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (20 mmol) and Streptomycin (401) (20 mmol).After 2 hours the reaction mixture is cooled in an ice water bath andtreated further with sodium cyanoborohydride (4.0 mmol) andtrifluoroacetic acid (30 mmol). After 2 additional hours the crudeproduct is precipitated by dropwise addition to a ten-fold volume ofacetonitrile, and then fractionated by reverse-phase HPLC to afford thetitle product.

The chemistry is detailed below in the following reaction scheme:

Example 11

Preparation of (409), a Compound of Formula X Via Scheme J.

Streptomycin (401) (10 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (20 mmol) and Fmoc glycinal (101) (10 mmol)(prepared as described by Salvi et al. Tetrahedron Lett. 1994, 35,1181–1184). After 2 hours the reaction mixture is cooled in an ice waterbath and treated further with sodium cyanoborohydride (4.0 mmol) andtrifluoroacetic acid (30 mmol). After 2 additional hours the crudeproduct is precipitated by dropwise addition to a ten-fold volume ofacetonitrile, and then fractionated by reverse-phase HPLC to afford thedesired product (407).

The above product (407) is then dissolved in anhydrous dimethylformamide(10 mL), stirred at room temperature and treated with excess piperidine(1.0 mL). After one hour the crude products are precipitated by dropwiseaddition to 100 mL acetonitrile with vigorous stirring. The crudeproducts are fractionated by reverse-phase HPLC to afford the desiredproduct (408).

Compound (408) (20 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (20 mmol) and Streptomycin (401) (20 mmol).After 2 hours the reaction mixture is cooled in an ice water bath andtreated further with sodium cyanoborohydride (4.0 mmol) andtrifluoroacetic acid (30 mmol). After 2 additional hours the crudeproduct is precipitated by dropwise addition to a ten-fold volume ofacetonitrile, and then fractionated by reverse-phase HPLC to afford thetitle product.

The chemistry is detailed below in the following reaction scheme:

Example 12

Preparation of (504), a Compound of Formula XI Via Scheme K.

Tetracycline (501) (10 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (20 mmol) and Fmoc glycinal (101) (10 mmol)(prepared as described by Salvi et al. Tetrahedron Lett. 1994, 35,1181–1184). After 2 hours the reaction mixture is cooled in an ice waterbath and treated further with sodium cyanoborohydride (4.0 mmol) andtrifluoroacetic acid (30 mmol). After 2 additional hours the crudeproduct is concentrated under reduced pressure and then fractionated byreverse-phase HPLC to afford the desired product (502).

The above product (502) is then dissolved in anhydrous dimethylformamide(10 mL), stirred at room temperature and treated with excess piperidine(1.0 mL). After one hour the crude product is concentrated under reducedpressure and then fractionated by reverse-phase HPLC to afford thedesired product (503).

Adipic acid (205) (2.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated diacidis treated with compound (503) (4.0 mmol) and the coupling reactionmixture is stirred overnight at room temperature. Volatiles are removedunder vacuum and the crude is fractionated by reverse-phase HPLC toafford the title product after lyopholization of the appropriatefractions.

Compound (501) is reported in Can. J. Chem. 1965, 43, 1382–1388; CAS1771-31-9, 53864-51-0

The chemistry is detailed below in the following reaction scheme:

Example 13

Preparation of (507), a Compound of Formula XII Via Scheme L.

Compound (505) (1.0 mmol) is dissolved in toluene, stirred in anice/water bath, and treated sequentially with K₂CO₃ (10 mmol) andcarbonyldiimidazole (1.0 mmol). The ice bath is removed and the reactionmixture is allowed to warm to room temperature. The imidazolide (506)thus produced is used without further manipulation in the couplingreactions described below.

A solution of (506) (1.0 mmol) in toluene/DMF with 1,4-diaminobutane(106) (0.5 mmol) is heated as necessary in a sealed vessel and thereaction followed by TLC. When judged complete, volatiles are removedunder vacuum and the crude is fractionated by reverse-phase HPLC toafford the title product after lyopholization of the appropriatefractions.

Compound (505) is reported in J. Med. Chem. 1994, 37, 3205–3211.

The chemistry is detailed below in the following reaction scheme:

Example 14

Preparation of (509), a Compound of Formula XIII Via Scheme M.

A solution of (506) (6 mmol), prepared as described in Example 13, intoluene/DMF with 6-aminocaproic acid (109) (6 mmol) is heated asnecessary in a sealed vessel and the reaction followed by TLC. Whenjudged complete, volatiles are removed under vacuum and the crude isfractioned by reverse-phase HPLC to afford the desired product (508)after lyopholization of the appropriate fractions.

Compound (508) (4.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated with compound (503) (4.0 mmol), preparedas described in Example 12, hydroxybenzotriazole (5.0 mmol),diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol) and the couplingreaction mixture is stirred overnight at room temperature. Volatiles areremoved under vacuum and the crude is fractioned by reverse-phase HPLCto afford the title product after lyopholization of the appropriatefractions.

The chemistry is detailed below in the following reaction scheme:

Example 15

Preparation of (605), a Compound of Formula XIV Via Scheme N.

Pristinamycin I (601) (10 mmol) is dissolved in MeOH, stirred at roomtemperature and treated sequentially with formalin (11 mmol), morpholine(11 mmol) and methanesulfonic acid (1 mmol), and then heated to 40° C.for 8 hours. The reaction mixture is partitioned between saturatedsodium bicarbonate solution and ethyl acetate. The organic phase isdried over anhydrous sodium sulfate, and volatiles removed under reducedpressure to afford crude morpholine adduct (602) which is used withoutfurther purification.

Compound (602) is dissolved in ethyl acetate, treated with a mixture ofsodium acetate and acetic acid, and heated to 45° C. for 4 hours. Thereaction mixture is then washed with sodium bicarbonate solution, dried,filtered, evaporated, and purified by chromatography to afford thedesired product (603).

Piperazine (5.0 mmol) is dissolved in 10 mL anhydrous tetrahydrofuranand stirred under nitrogen in a dry ice/acetone bath. A solution of 1.0M butyl lithium (11 mmol) is added via syringe, and the mixture isstirred for 15 minutes. At this point a solution of ethylene sulfide (11mmol) in 1 mL anhydrous tetrahydrofuran is added dropwise via syringeover 5 minutes. The reaction mixture is then removed from the coolingbath and allowed to slowly rise to room temperature, where it is stirredfor 2 hours. The reaction mixture is then quenched with ammoniumchloride. Volatiles are then removed under reduced pressure and thecrude product is fractionated by silica gel chromatography to affordpiperazine thiol (604). A solution of 10 mmols of (603) in acetone with5 mmols of (604) in acetone is cooled to −20° C. and the reactionfollowed by TLC. When judged complete, volatiles are removed undervacuum and the crude is fractionated by reverse-phase HPLC to afford thetitle product after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 16

Preparation of (608), a Compound of Formula XV Via Scheme O.

To a solution of Quinupristin (606) (5 mmol) in water acidified to pH 2with HCl is added zinc (40 mmol). The reaction mixture is filtered andthe filtrate is diluted with Na₂CO₃, extracted with CH₂Cl₂, dried overMgSO₄ and concentrated. The crude product is purified by chromatographyover silica gel to afford compound (607).

Adipic acid (205) (2.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated diacidis treated with compound (607) (4.0 mmol) and the coupling reactionmixture is stirred overnight at room temperature. Volatiles are removedunder vacuum and the crude is fractionated by reverse-phase HPLC toafford the title product after lyopholization of the appropriatefractions.

The chemistry is detailed below in the following reaction scheme:

Example 17

Preparation of (611), a Compound of Formula XVI Via Scheme P.

A solution of 20 mmols of (603), prepared as described in Example 15, inacetone with 20 mmols of 3-mercaptopropionic acid (609) is cooled to−20° C. and the reaction followed by TLC. When judged complete,volatiles are removed under vacuum and the crude product is purified bychromatography to afford the desired product (610).

Compound (610) (4.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with compound (607) (4.0mmol), prepared as described in Example 16, hydroxybenzotriazole (5.0mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol) and thecoupling reaction mixture is stirred overnight at room temperature.Volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the title product after lyopholization ofthe appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 18

Preparation of (613), a Compound of Formula XVII Via Scheme Q.

A solution of 20 mmols of Pristinamycin II (612) in acetone with 10mmols of (604), prepared as described in Example 15 is cooled to −20° C.and the reaction followed by TLC. When judged complete, the mixture ispartitioned between ethyl acetate and water and the organic phase washedwith water, dried over sodium sulfate and the solvent removed in vacuo.The residue is purified by chromatography to afford the title structure.

The chemistry is detailed below in the following reaction scheme:

Example 19

Preparation of (616), a Compound of Formula XVIII Via Scheme R.

A solution of 20 mmols of (603) in acetone with 20 mmols of1,6-hexanedithiol (614) is cooled to −20° C. and the reaction followedby TLC. When judged complete, volatiles are removed under vacuum and thecrude is fractionated by reverse-phase HPLC to afford the desiredproduct (615) after lyopholization of the appropriate fractions.

A solution of 20 mmols of (615) in acetone with 20 mmols ofPristinamycin II (612) is cooled to −20° C. and the reaction followed byTLC. When judged complete, volatiles are removed under vacuum and thecrude is fractionated by reverse-phase HPLC to afford the title productafter lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 20

Preparation of (618), a Compound of Formula XIX Via Scheme S.

A solution of 20 mmols of Pristinamycin II (612) in acetone with 20mmols of 3-mercaptopropionic acid (609) is cooled to −20° C. and thereaction followed by TLC. When judged complete, volatiles are removedunder vacuum and the crude is fractionated by reverse-phase HPLC toafford the desired product (617) after lyopholization of the appropriatefractions.

Compound (617) (4.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated with compound (607) (4.0 mmol), preparedas described in Example 16, hydroxybenzotriazole (5.0 mmol),diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol) and the couplingreaction mixture is stirred overnight at room temperature. Volatiles areremoved under vacuum and the crude is fractionated by reverse-phase HPLCto afford the title product after lyopholization of the appropriatefractions.

The chemistry is detailed below in the following reaction scheme:

Example 21

Preparation of (702), (703), and (704), Compounds of Formula XX ViaScheme T.

A solution of 20 mmols of Spectinomycin (701) in DMF with 10 mmols ofα,α′-dibromo-p-xylene and 40 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When judged complete,volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the title products after lyopholization ofthe appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 22

Preparation of (806), (807), and (808).

Gentamicin C₁ (801) (10 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (60 mmol) and benzaldehyde (50 mmol). After2 hours the reaction mixture is cooled in an ice water bath and treatedfurther with sodium cyanoborohydride (20 mmol) and trifluoroacetic acid(70 mmol). After 2 additional hours the crude product is precipitated bydropwise addition to a ten-fold volume of acetonitrile, and thenfractionated by reverse-phase HPLC to afford the desired product (802).

Compound (802) (10 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (20 mmol) and Fmoc glycinal (101) (10 mmol)(prepared as described by Salvi et al. Tetrahedron Lett. 1994, 35,1181–1184). After 2 hours the reaction mixture is cooled in an ice waterbath and treated further with sodium cyanoborohydride (4.0 mmol) andtrifluoroacetic acid (30 mmol). After 2 additional hours the crudeproduct is precipitated by dropwise addition to a ten-fold volume ofacetonitrile, and then fractionated by reverse-phase HPLC to afford thedesired products (803), (804), and (805).

The above separated products (803), (804), and (805) are thenindividually dissolved in anhydrous dimethylformamide (10 mL), stirredat room temperature and treated with excess piperidine (1.0 mL). Afterone hour, the crude products are concentrated under reduced pressure andthen fractionated by reverse-phase HPLC to afford compounds (806),(807), and (808),

The chemistry is detailed below in the following reaction scheme:

Example 23

Preparation of (803), (804), (805), (813), (814), (815), (816), and(817).

Gentamicin C₂ (809) (10 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (60 mmol) and benzaldehyde (50 mmol). After2 hours the reaction mixture is cooled in an ice water bath and treatedfurther with sodium cyanoborohydride (20 mmol) and trifluoroacetic acid(70 mmol). After 2 additional hours the crude product is precipitated bydropwise addition to a ten-fold volume of acetonitrile, and thenfractionated by reverse-phase HPLC to afford the desired product (811).Compound (812) is prepared in the same manner from Gentamicin C₃ (810).

Compound (811) (10 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (20 mmol) and Fmoc glycinal (101) (10 mmol)(prepared as described by Salvi et al. Tetrahedron Lett. 1994, 35,1181–1184). After 2 hours the reaction mixture is cooled in an ice waterbath and treated further with sodium cyanoborohydride (4.0 mmol) andtrifluoroacetic acid (30 mmol). After 2 additional hours the crudeproduct is precipitated by dropwise addition to a ten-fold volume ofacetonitrile, and then fractionated by reverse-phase HPLC to afford thedesired products (803), (804), (805), and (813), which are thenseparated by reverse phase chromatography. Compounds (814), (815),(816), and (817) are prepared and separated in the same manner from(812).

The above separated products (803), (804), (805), and (813) are thenindividually dissolved in anhydrous dimethylformamide (10 mL), stirredat room temperature and treated with excess piperidine (1.0 mL). Afterone hour, the crude products are fractionated by reverse-phase HPLC toafford compounds (806), (807), (808), and (818). Compounds (820), (821),and (822) are prepared and separated in the same manner from (814),(815), (816), and (817), respectively.

The chemistry is detailed below in the following reaction scheme:

Example 24

Preparation of (824), a Compound of Formula XXI Via Scheme U.

Compound (806) (10 mmol), prepared as described in Example 22, isslurried in methanol:anhydrous dimethylformamide, stirred at roomtemperature, and treated sequentially with diisopropylethyl amine (20mmol) and Streptomycin (401) (10 mmol). After 2 hours the reactionmixture is cooled in an ice water bath and treated further with sodiumcyanoborohydride (4.0 mmol) and trifluoroacetic acid (30 mmol). After 2additional hours the crude product is precipitated by dropwise additionto a ten-fold volume of acetonitrile, and then fractionated byreverse-phase HPLC to afford the desired product (823).

A solution of (823) (5 mmol) in methanol is hydrogenated overnight at 35psi, in the presence of 10% Pd/C. When judged completes volatiles areremoved under vacuum and the crude is fractionated by reverse-phase HPLCto afford the title product after lyopholization of the appropriatefractions.

NOTE: Compound (806) is shown in the example, but the same procedure canbe carried out on compounds (807), (808), (818), (819), (820), (821),(822), and (829) to provide their respective dimers with streptomycin.

The chemistry is detailed below in the following reaction scheme:

Example 25

Preparation of (828), a Compound of Formula XXII Via Scheme V.

Terephthalic acid (876) (2.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated diacidis treated with compound (806) (4.0 mmol), prepared as described inExample 22, and the coupling reaction mixture is stirred overnight atroom temperature. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the desired product (827)after lyopholization of the appropriate fractions.

A solution of (827) (5 mmol) in methanol is hydrogenated overnight at 35psi, in the presence of 10% Pd/C. When judged complete, volatiles areremoved under vacuum and the crude is fractionated by reverse-phase HPLCto afford the title product after lyopholization of the appropriatefractions.

NOTE: Compound (806) is shown in the example, but the same procedure canbe carried out on compounds (807), (808), (818), (819), (820), (821),and (822) to provide their respective dimers.

The chemistry is detailed below in the following reaction scheme:

Example 26

Preparation of (830), a Compound of Formula XXIII Via Scheme W.

A solution of 20 mmols of (811), prepared as described in Example 23, inTHF with 80 mmols of triethylamine is treated at room temperature with40 mmols of trifluoroacetic anhydride for 1 hour. A solution of theresulting product (11.7 mmol) in methanol is hydrogenated overnight at35 psi, in the presence of 10% Pd/C. The reaction mixture is filteredand the filtrate is concentrated to dryness. The crude product ispurified by reverse-phase HPLC to afford the desired compound (829).

A solution of 20 mmols of compound (829) in DMF with 10 mmols of1,6-dibromohexane (302) and 40 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When judged complete,volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the title product after lyopholization ofthe appropriate fractions.

NOTE: Compound (811) is shown in the example, but the same procedure canbe carried out on compounds (802) and (812) to provide the respectiveproducts.

The chemistry is detailed below in the following reaction scheme:

Example 27

Preparation of (831), a Compound of Formula XXIV Via Scheme X.

A solution of 20 mmols of compound (829), prepared as described inExample 26, in DMF with 20 mmols of 3-bromopropionic acid (833) and 200mmols of potassium carbonate is heated as necessary and the reactionfollowed by TLC. When judged complete, volatiles are removed undervacuum and the crude is fractionated by reverse-phase HPLC to afford thedesired product (834) after lyopholization of the appropriate fractions.

Compound (834) (4.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated acid istreated with compound (806) (4.0 mmol) and the coupling reaction mixtureis stirred overnight at room temperature. The mixture is dilutedtwo-fold with an aqueous NaOH solution (25 mmol) and heated to 50° C.for 1 hour. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the title product afterlyopholization of the appropriate fractions.

NOTE: Compound (806) is shown in the example, but the same procedure canbe carried out on compounds (807), (808), (818), (819), (820), (821),and (822) to provide their respective dimers with compound (829).

The chemistry is detailed below in the following reaction scheme:

Example 28

Preparation of (832), a Compound of Formula XXV Via Scheme Y.

Succinic acid (104) (2.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(2.0 mmol), diisopropylethyl amine (2.0 mmol) and PyBOP (2.0 mmol).After stirring for 15 minutes at room temperature, the activated diacidis treated with compound (408) (2.0 mmol), prepared as described inExample 11, and the coupling reaction mixture is stirred overnight atroom temperature. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the desired product (835)after lyopholization of the appropriate fractions.

Compound (835) (4.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated diacidis treated with compound (806) (4.0 mmol), prepared as described inExample 22, and the coupling reaction mixture is stirred overnight atroom temperature. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the title product afterlyopholization of the appropriate fractions. A solution of the resultingproduct (11.7 mmol) in methanol is hydrogenated overnight at 35 psi, inthe presence of 10% Pd/C. The reaction mixture is filtered and thefiltrate is concentrated to dryness. The crude product is purified byreverse-phase HPLC to afford the title product.

NOTE: Compound (806) is shown in the example, but the same procedure canbe carried out on compounds (807), (808), (818), (819), (820), (821),and (822) to provide their respective dimers with compound (408).

The chemistry is detailed below in the following reaction scheme:

Example 29

Preparation of (902), a Compound of Formula XXVI Via Scheme Z.

A solution of 10 mmols of compound (301) in DMF with 10 mmols of3-bromopriopionic acid (833) and 20 mmols of potassium carbonate isheated as necessary and the reaction followed by TLC. When judgedcomplete, volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the desired product (901)after lyopholization of the appropriate fractions.

Compound (901) (4.0 mmol) is dissolved in anhydrous dimethylformamideand treated sequentially with hydroxybenzotriazole (5.0 mmol),diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol). After stirringfor 15 minutes at room temperature, the activated acid is treated withcompound (103) (4.0 mmol), prepared as described in Example 1, and thecoupling reaction mixture is stirred overnight at room temperature.Volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the title product after lyopholization ofthe appropriate fractions.

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother. 21:902–905 (19892).

The chemistry is detailed below in the following reaction scheme:

Example 30

Preparation of (905), a Compound of Formula XXVII Via Scheme AA.

1,4-Diaminobutane (106) (4.0 mmol) is dissolved in DMF, stirred at roomtemperature, and treated sequentially with diisopropylethyl amine (4.0mmol) and imidazolide (105) (4.0 mmol), prepared as described in Example2. After 2 hours, volatiles are removed under vacuum and the crudeproduct is purified by reverse-phase HPLC to afford the desired product(903).

Compound (903) (2.0 mmol) is dissolved in DMF, stirred at roomtemperature, and treated sequentially with diisopropylethyl amine (4.0mmol) and imidazolide (202) (2.0 mmol), prepared as described in Example4. After 2 hours, volatiles are removed under vacuum and the crudeproduct is purified by reverse-phase HPLC to afford the desired product(904).

Compound (904) (2.0 mmol) is dissolved in THF. Trimethylsilyl triflate(20 mmol) and lutidine (30 mmol) are added and the reaction is followedby TLC. When judged complete, the mixture is treated withtetrabutylammonium fluoride (30 mmol) and the reaction is followed byTLC. When judged complete, the mixture is diluted two-fold with methanoland heated at refux for 1 hour. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 31

Preparation of (905), a Compound of Formula XXVIII Via Scheme BB.

Succinic acid (104) (8.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole (10mmol), diisopropylethyl amine (8.0 mmol) and PyBOP (8.0 mmol). Afterstirring for 15 minutes at room temperature, the diacid is treated withcompound (103) (8.0 mmol), prepared as described in Example 1, and thecoupling reaction mixture is stirred overnight at room temperature.Volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the desired product (906) afterlyopholization of the appropriate fractions.

Compound (906) (2.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(2.5 mmol), diisopropylethyl amine (2.0 mmol) and PyBOP (2.0 mmol).After stirring for 15 minutes at room temperature, the activated acid istreated with compound (204) (2.0 mmol) and the coupling reaction mixtureis stirred overnight at room temperature. Volatiles are removed undervacuum and the crude is fractionated by reverse-phase HPLC to afford thetitle product after lyopholization of the appropriate fractions.

Compound (204) is reported in J. Med. Chem. 1996, 49, 673–679.

The chemistry is detailed below in the following reaction scheme:

Example 32

Preparation of (909), a Compound of Formula XXIX Via Scheme CC.

Compound (110) (4.0 mmol), prepared as described in Example 3, isdissolved in anhydrous dimethylformamide and treated sequentially withhydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol). After stirring for 15 minutes at room temperature, theactivated acid is treated with compound (204) (4.0 mmol) and thecoupling reaction mixture is stirred overnight at room temperature.Volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the desired product (908) afterlyopholization of the appropriate fractions.

Compound (908) (2.0 mmol) is dissolved in THF. Trimethylsilyl triflate(20 mmol) and lutidine (30 mmol) are added and the reaction is followedby TLC. When judged complete, the mixture is treated withtetrabutylammonium fluoride (30 mmol) and the reaction is followed byTLC. When judged complete, the mixture is diluted two-fold with methanoland heated at reflux for 1 hour. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

Compound (204) is reported in J. Med. Chem. 1996, 49, 673–679.

The chemistry is detailed below in the following reaction scheme:

Example 33

Preparation of (911), a Compound of Formula XXX Via Scheme DD.

A solution of (105) (2.0 mmol), prepared as described in Example 2, intoluene/DMF with 1,4-diaminobutane (106) (2.0 mmol) is heated asnecessary in a sealed vessel and the reaction followed by TLC. Whenjudged complete, volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the desired product (922)after lyopholization of the appropriate fractions.

A solution of the above product (922) (1.0 mmol) in DMF with compound(202) (1.0 mmol), prepared as described in Example 4, is heated asnecessary in a sealed vessel and the reaction followed by TLC. Whenjudged complete, volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the desired product (910)after lyopholization of the appropriate fractions.

Compound (910) (2.0 mmol) is dissolved in THF. Trimethylsilyl triflate(20 mmol) and lutidine (30 mmol) are added and the reaction is followedby TLC. When judged complete, the mixture is treated withtetrabutylammonium fluoride (30 mmol) and the reaction is followed byTLC. When judged complete, the mixture is diluted two-fold with methanoland heated at reflux for 1 hour. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 34

Preparation of (923) and (924), Compounds of Formula XXXI Via Scheme EE.

A solution of 10 mmols of Spectinomycin (701) in DMF with 10 mmols of3-bromopropionic acid (833) and 20 mmols of potassium carbonate isheated as necessary and the reaction followed by TLC. When judgedcomplete, volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the desired products (912)and (913) after lyopholization of the appropriate fractions.

Compounds (912) (4.0 mmol) and (913) (4.0 mmol) are individuallydissolved in anhydrous dimethylformamide and treated sequentially withcompound (103) (4.0 mmol), prepared as described in Example 1,hydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol) and the coupling reaction mixture is stirred overnightat room temperature. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the title products afterlyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 35

Preparation of (915), a Compound of Formula XXXII Via Scheme FF.

1,4-Diaminobutane (106) (4.0 mmol) is dissolved in toluene/DMF, stirredat room temperature, and treated sequentially with diisopropylethylamine (4.0 mmol) and imidazolide (105) (4.0 mmol), prepared as describedin Example 2. After 2 hours, volatiles are removed under vacuum and thecrude is fractionated by reverse-phase HPLC to afford the desiredproduct (903) after lyopholization of the appropriate fractions.

Compound (903) (2.0 mmol) is dissolved in toluene, stirred at roomtemperature, and treated sequentially with diisopropylethyl amine (4.0mmol) and imidazolide (506) (2.0 mmol), prepared as described in Example13. After 2 hours, volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the desired product (914)after lyopholization of the appropriate fractions.

Compound (914) (2.0 mmol) is dissolved in THF. Trimethylsilyl triflate(20 mmol) and lutidine (30 mmol) are added and the reaction is followedby TLC. When judged complete, the mixture is treated withtetrabutylammonium fluoride (30 mmol) and the reaction is followed byTLC. When judged complete, the mixture is diluted two-fold with methanoland heated at reflux for 1 hour. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 36

Preparation of (916), a Compound of Formula XXXIII Via Scheme GG.

Compound (906) (2.0 mmol), prepared as described in Example 31, isdissolved in 10 mL anhydrous dimethylformamide and treated sequentiallywith hydroxybenzotriazole (2.5 mmol), diisopropylethyl amine (2.0 mmol)and PyBOP (2.0 mmol). After stinting for 15 minutes at room temperature,the activated acid is treated with compound (503) (2.0 mmol), preparedas described in Example 12, and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 37

Preparation of (918), a Compound of Formula XXXIV Via Scheme HH.

Compound (110) (4.0 mmol), prepared as described in Example 3, isdissolved in anhydrous dimethylformamide and treated sequentially withhydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol). After stirring for 15 minutes at room temperature, theactivated acid is treated with compound (503) (4.0 mmol), prepared asdescribed in Example 12, and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the desiredproduct (908) after lyopholization of the appropriate fractions.

Compound (908) (2.0 mmol) is dissolved in THF. Trimethylsilyl triflate(20 mmol) and lutidine (30 mmol) are added and the reaction is followedby TLC. When judged complete, the mixture is treated withtetrabutylammonium fluoride (30 mmol) and the reaction is followed byTLC. When judged complete, the mixture is diluted two-fold with methanoland heated at reflux for 1 hour. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 38

Preparation of (921), a Compound of Formula XXXV Via Scheme II.

A solution of compound (922) (1.0 mmol), prepared as described inExample 33, in DMF with compound (506) (1.0 mmol), prepared as describedin Example 13, is heated as necessary in a sealed vessel and thereaction followed by TLC. When judged complete, volatiles are removedunder vacuum and the crude is fractionated by reverse-phase HPLC toafford the desired product (920) after lyopholization of the appropriatefractions.

Compound (920) (2.0 mmol) is dissolved in THF. Trimethylsilyl triflate(20 mmol) and lutidine (30 mmol) are added and the reaction is followedby TLC. When judged complete, the mixture is treated withtetrabutylammonium fluoride (30 mmol) and the reaction is followed byTLC. When judged complete, the mixture is diluted two-fold with methanoland heated at reflux for 1 hour. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 39

Preparation of (1001), a Compound of Formula XXXVI Via Scheme JJ.

3-Bromopropionic acid (833) (4.0 mmol) is dissolved in anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated acid istreated with compound (204) (4.0 mmol) and the coupling reaction mixtureis stirred overnight at room temperature. Volatiles are removed undervacuum and the crude product is purified by silica gel chromatography toafford the desired product (1000).

A solution of 2 mmols of the above compound (1000) in DMF with 2 mmolscompound (301) and 20 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC, When judged complete,volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the title product after lyopholization ofthe appropriate fractions.

Compound (204) is reported in J. Med. Chem., 49:673–679 (1996).

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother., 21:902–905 (1982).

The chemistry is detailed below in the following reaction scheme:

Example 39A

Preparation of (1012), a Compound of Formula XXXVII Via Scheme KK.

A solution of (202) (20 mmol), prepared as described in Example 4, inDMF with 3-bromopropylamine hydrobromide (1011) (20 mmol) is healed asnecessary in a sealed vessel and the reaction followed by TLC. Whenjudged complete, the mixture is partitioned between ethyl acetate andwater and the organic phase washed with water, dried over sodium sulfateand the solvent removed in vacuo. The residue is purified bychromatography to afford the desired product (1002).

A solution of 10 mmols of the above compound (1002) in DMF with 10 mmolsof compound (301) and 20 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When judged complete,volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the title product after lyopholization ofthe appropriate fractions.

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother. 1982, 21, 902–905.

The chemistry is detailed below in the following reaction scheme:

Example 40

Preparation of (1004), a Compound of Formula XXXVIII Via Scheme LL.

A solution of 20 mmols of the compound (301) in DMF with 20 mmols3-bromoropionic acid (833) and 40 mmols of potassium carbonate is heatedas necessary and the reaction followed by TLC. When judged complete, themixture is partitioned between ethyl acetate and water and the organicphase washed with water, dried over sodium sulfate and the solventremoved in vacuo. The residue is purified by chromatography to affordthe desired product (1003).

The above product (1003) (4.0 mmol) is dissolved in anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated acid istreated with compound (503) (4.0 mmol), prepared as described in Example12, and the coupling reaction mixture is stirred overnight at roomtemperature. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the title product afterlyopholization of the appropriate fractions.

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother. 1982, 21, 902–905.

The chemistry is detailed below in the following reaction scheme:

Example 41

Preparation of (1006) and (1007), Compounds of Formula XXXIX Via SchemeMM.

A solution of 20 mmols of compound (301) in DMF with 20 mmols of1,6-diaminohexane (302) and 40 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When judged complete,volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the desired product (1005) afterlyopholization of the appropriate fractions.

A solution of 10 mmols of the above compound (1005) in DMF with 10 mmolsof Spectinomycin (701) and 20 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When judged complete,volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the title product after lyopholization ofthe appropriate fractions.

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother. 1982, 21, 902–905.

Example 42

Preparation of (1010), a Compound of Formula XL Via Scheme NN.

A solution of 20 mmols of 6-bromohexanoic acid (1008) in DMF with 20mmols of compound (301) and 40 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When judged complete, thereaction mixture is concentrated under reduced pressure and the residuepurified by chromatography to afford the desired product (1009).

The above compound (1009) (4.0 mmol) is dissolved in anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated acid istreated with compound (503) (4.0 mmol), prepared as described in Example12, and the coupling reaction mixture is stirred overnight at roomtemperature. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the title product afterlyopholization of the appropriate fractions.

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother. 1982, 21, 902–905.

The chemistry is detailed below in the following reaction scheme:

Example 43

Preparation of (1101), a Compound of Formula XLI Via Scheme OO.

Compound (906) (4.0 mmol), prepared as described in Example 31, isdissolved in anhydrous dimethylformamide and treated sequentially withhydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol). After stirring for 15 minutes at room temperature, theactivated acid is treated with compound (607) (4.0 mmol), prepared asdescribed in Example 16, and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 44

Preparation of (1102), a Compound of Formula XLII Via Scheme PP.

Compound (610) (4.0 mmol), prepared as described in Example 17, isdissolved in anhydrous dimethylformamide and treated sequentially withhydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol). After stirring for 15 minutes at room temperature, theactivated acid is treated with compound (103) (4.0 mmol), prepared asdescribed in Example 1, and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 45

Preparation of (1103), a Compound of Formula XLIII Via Scheme QQ.

Compound (617) (4.0 mmol), prepared as described in Example 16, isdissolved in anhydrous dimethylformamide and treated sequentially withhydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol). After stirring for 15 minutes at room temperature, theactivated acid is treated with compound (103) (4.0 mmol), prepared asdescribed in Example 1, and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 46

Preparation of (1105), a Compound of Formula XLIV Via Scheme RR.

Compound (901) (4.0 mmol), prepared as described in Example 29, isdissolved in anhydrous dimethylformamide and treated sequentially withhydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol). After stirring for 15 minutes at room temperature, theactivated acid is treated with compound (607) (4.0 mmol), prepared asdescribed in Example 16, and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 47

Preparation of (1113), a Compound of Formula XLV Via Scheme SS.

Compound (301) (10 mmol) is slurried in methanol:anhydrousdimethylformamide, stirred at room temperature, and treated sequentiallywith diisopropylethyl amine (20 mmol) and Fmoc glycinal (101) (10 mmol)(prepared as described by Salvi et al. Tetrahedron Lett. 1994, 35,1181–1184). After 2 hours the reaction mixture is cooled in an ice waterbath and treated further with sodium cyanoborohydride (4.0 mmol) andtrifluoroacetic acid (30 mmol). After 2 additional hours, volatiles areremoved under vacuum and the crude is fractionated by reverse-phase HPLCto afford the desired product (1106) after lyopholization of theappropriate fractions.

The above product (1106) is then dissolved in anhydrousdimethylformamide (10 mL), stirred at room temperature and treated withexcess piperidine (1.0 mL). After one hour the crude products arefractionated by reverse-phase HPLC to afford the desired product (1112).

Compound (617) (4.0 mmol), prepared as described in Example 20, isdissolved in 10 mL anhydrous dimethylformamide and treated sequentiallywith hydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol)and PyBOP (4.0 mmol). After stirring for 15 minutes at room temperature,the activated acid is treated with compound (1112) (4.0 mmol) and thecoupling reaction mixture is stirred overnight at room temperature.Volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the title product after lyopholization ofthe appropriate fractions.

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother. 1982, 21, 902–905.

The chemistry is detailed below in the following reaction scheme:

Example 48

Preparation of(1108), a Compound of Formula XLVI Via Scheme TT.

Compound (204) (10 mmol) is dissolved in anhydrous dimethylformamide andtreated with adipic acid (205) (10 mmol), hydroxybenzotriazole (10mmol), diisopropylethyl amine (20 mmol) and PyBOP (10 mmol) and thecoupling reaction mixture is stirred overnight at room temperature.After deprotection with NaOH, volatiles are removed under vacuum and thecrude is fractionated by reverse-phase HPLC to afford the desiredproduct (1107) after lyopholization of the appropriate fractions.

The above compound (1107) (4.0 mmol) is dissolved in anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated acid istreated with compound (607) (4.0 mmol), prepared as described in Example16, and the coupling reaction mixture is stirred overnight at roomtemperature. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the title product afterlyopholization of the appropriate fractions.

Compound (204) is reported in J. Med. Chem. 1996, 49, 673–679.

The chemistry is detailed below in the following reaction scheme:

Example 49

Preparation of (1109), a Compound of Formula XLVII Via Scheme UU.

Compound (207) (4.0 mmol), prepared as described in Example 6, isdissolved in anhydrous dimethylformamide and treated sequentially withhydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol). After stirring for 15 minutes at room temperature, theactivated acid is treated with compound (607) (4.0 mmol), prepared asdescribed in Example 16, and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 50

Preparation of (1110), a Compound of Formula XLVIII Via Scheme VV.

Compound (610) (4.0 mmol), prepared as described in Example 17, isdissolved in anhydrous dimethylformamide and treated sequentially withhydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol). After stirring for 15 minutes at room temperature, theactivated acid is treated with compound (204) (4.0 mmol), prepared asdescribed in Example 16, and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

Compound (204) is reported in J. Med. Chem. 1996, 49, 673–679.

The chemistry is detailed below in the following reaction scheme:

Example 51

Preparation of (1111), a Compound of Formula XLIX Via Scheme WW.

Compound (617) (4.0 mmol), prepared as described in Example 20, isdissolved in anhydrous dimethylformamide and treated sequentially withhydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0 mmol) andPyBOP (4.0 mmol). After stirring for 15 minutes at room temperature, theactivated acid is treated with compound (204) (4.0 mmol), prepared asdescribed in Example 16, and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproduct after lyopholization of the appropriate fractions.

Compound (204) is reported in J. Med. Chem. 1996, 49, 673–679.

The chemistry is detailed below in the following reaction scheme:

Example 52

Preparation of (1201), a Compound of Formula L Via Scheme XX.

A solution of 20 mmols of compound (829), prepared as described inExample 26, in DMF with 20 mmols of 1,6-dibromohexane (302) and 40 mmolsof potassium carbonate is heated as necessary and the reaction followedby TLC. When judged complete, the crude product is purified bychromatography to afford the desired product (1200).

A solution of 10 mmols of the above compound (1200) in DMF with 10 mmolsof compound (301) and 20 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When judged complete, thereaction mixture is diluted two-fold with water, treated with NaOH (50mmol), and heated as necessary to effect deprotection. The crude productis fractionated by reverse-phase HPLC to afford the title product afterlyopholization of the appropriate fractions.

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother. 1982, 21, 902–905.

The chemistry is detailed below in the following reaction scheme:

Example 53

Preparation of (1202) and (1203), Compounds of Formula LI Via Scheme YY.

A solution of 20 mmols of the compound (1200), prepared as described inExample 52, in DMF with 20 mmols of Spectinomycin (701) and 20 mmols ofpotassium carbonate is heated as necessary and the reaction followed byTLC. When judged complete, the reaction mixture is diluted two-fold withwater, treated with NaOH (50 mmol), and heated as necessary to effectdeprotection. The crude product is fractionated by reverse-phase HPLC toafford the title product after lyopholization of the appropriatefractions.

The chemistry is detailed below in the following reaction scheme:

Example 54

Preparation of (1205) and (1206), Compounds of Formula LII Via SchemeZZ.

A solution of 20 mmols of compound (301) in DMF with 20 mmols of1,6-dibromohexane (302) and 40 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When judged complete,volatiles are removed under reduced pressure to afford the desiredproduct (1204), which is used in the next step without any furtherpurification.

A solution of 10 mmols of the above compound (1204) in DMF with 10 mmolsof Spectinomycin (701) and 20 mmols of potassium carbonate is heated asnecessary and the reaction followed by TLC. When judged complete,volatiles are removed under vacuum and the crude is fractionated byreverse-phase HPLC to afford the title product after lyopholization ofthe appropriate fractions.

Compound (301) is U-57930E and is reported in Antimicrobial AgentsChemother. 1982, 21, 902–905.

The chemistry is detailed below in the following reaction scheme:

Example 55

Preparation of (1207) and (1208), Compounds of Formula LIII Via SchemeAAA.

Compounds (912) (4.0 mmol) and (913) (4.0 mmol), prepared as describedin Example 34, are individually dissolved in anhydrous dimethylformamideand treated with compound (408) (4.0 mmol), prepared as described inExample 11, hydroxybenzotriazole (5.0 mmol), diisopropylethyl amine (4.0mmol) and PyBOP (4.0 mmol) and the coupling reaction mixture is stirredovernight at room temperature. Volatiles are removed under vacuum andthe crude is fractionated by reverse-phase HPLC to afford the titleproducts after lyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 56

Preparation of (1211), a Compound of Formula LIV Via Scheme BBB.

In a sealed tube, 1,3-phenylenediisocyanate (1209) (41.0 mmol) isdissolved in anhydrous acetonitrile. To this solution is added compound(408) (41.0 mmol), prepared as described in Example 11, and the tube ispartially immersed in a silicon oil bath and heated to 65° C. andallowed to stir for 16 h. The reaction mixture is cooled and thenevaporated to afford the crude product (1210), which is used in the nextstep without any further purification.

In a sealed tube, compound (1210) (30.0 mmol) is dissolved in anhydrousacetonitrile. To this solution is added compound (503) (30.0 mmol),prepared as described in Example 12, and the tube is partially immersedin a silicon oil bath and heated to 85° C. and allowed to stir for 16 h.The reaction mixture is cooled and then evaporated to afford the crudeproduct, which is purified by reverse-phase HPLC to afford the titleproduct

The chemistry is detailed below in the following reaction scheme:

Example 56A

Preparation of (1213), a Compound of Formula LV Via Scheme CCC.

Succinic acid (104) (8.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(10.0 mmol), diisopropylethyl amine (8.0 mmol) and PyBOP (8.0 mmol).After stirring for 15 minutes at room temperature, the activated acid istreated with compound (835) (8.0 mmol), prepared as described in Example28, and the coupling reaction mixture is stirred overnight at roomtemperature. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the desired product (1212)after lyopholization of the appropriate fractions.

The above compound (1212) (4.0 mmol) is dissolved in 10 mL anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated acid istreated with compound (503) (4.0 mmol), prepared as described in Example12, and the coupling reaction mixture is stirred overnight at roomtemperature. Volatiles are removed under vacuum and the crude isfractionated by reverse-phase HPLC to afford the title product afterlyopholization of the appropriate fractions.

The chemistry is detailed below in the following reaction scheme:

Example 57

Preparation of (1215), a Compound of Formula LVI Via Scheme DDD.

A solution of 10 mmols of compound (829), prepared as described inExample 26, in DMF with 10 mmols of 6-bromohexanoic acid (1008) and 20mmols of potassium carbonate is heated as necessary and the reactionfollowed by TLC. When judged complete, the reaction mixture isconcentrated under reduced pressure and the residue purified bychromatography to afford the desired product (1214).

The above compound (1214) (4.0 mmol) is dissolved in anhydrousdimethylformamide and treated sequentially with hydroxybenzotriazole(5.0 mmol), diisopropylethyl amine (4.0 mmol) and PyBOP (4.0 mmol).After stirring for 15 minutes at room temperature, the activated acid istreated with compound (503) (4.0 mmol), prepared as described in Example12, and the coupling reaction mixture is stirred overnight at roomtemperature. The reaction mixture is diluted two-fold with water,treated with NaOH (50 mmol), and heated as necessary to effectdeprotection. The crude product is fractionated by reverse-phase HPLC toafford the title product after lyopholization of the appropriatefractions.

The chemistry is detailed below in the following reaction scheme:

1. A compound of the formula:L′-X′-L″ or a pharmaceutically-acceptable salt thereof; wherein L′ is amoiety of the formula:

L″ is a moiety of the formula:

X′ has the formula:—X^(a)Z-(Y^(a)-Z)_(m)-Y^(b)-Z-X^(a)— wherein m is an integer of from 0to 20; X^(a) at each separate occurrence is selected from the groupconsisting of —O—, —S—, —NR—, —C(O)—, —C(O)O—, —C(O)NR—, —C(S), —C(S)O—,—C(S)NR— and a covalent bond, where R is as defined below; Z at eachseparate occurrence is selected from the group consisting of alkylene,substituted alkylene, cycloalkylene, substituted cylcoalkylene,alkenylene, substituted alkenylene, alkynylene, substituted alkynylene,cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene,heterocyclene, and a covalent bond; Y^(a) and Y^(b) at eaeh separateoccurrence are selected from the group consisting of —C(O)NR′—,—NR′C(O)—, —NR′C(O)NR′—, —C(═NR′)—NR′—, —NR′—C(═NR′)—, —NR′—C(O)O—,—P(O)(OR′)—O—, —S(O)_(n)CR′R″—, —S(O)_(n)NR′—, —S—S— and a covalentbond; where n is 0, 1 or 2 and R, R′ and R″ at each separate occurrenceare selected from the group consisting of hydrogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,aryl, heteroaryl and heterocyclic.
 2. The compound of claim 1, whereinL′ is formula (i).
 3. The compound of claim 1, where L′ is formula (ii).4. The compound of claim 1, wherein L″ is formula (iii).
 5. The compoundof claim 1, wherein L″ is formula (iv).
 6. A compound of the formula:L′-X′-L″ or a pharmaceutically-acceptable salt thereof; wherein L′ is amoiety of the formula:

L″ is a moiety of the formula:

X′ has the formula:—X^(a)-Z-(Y^(a)-Z)_(m)-Y^(b)-Z-X^(a)— wherein m is an integer of from 0to 20; X^(a) at each separate occurrence is selected from the groupconsisting of —O—, —S—, —NR—, —C(O)—, —C(O)O—, —C(O)NR—, —C(S), —C(S)O—,—C(S)NR— and a covalent bond, where R is as defined below; Z at eachseparate occurrence is selected from the group consisting of alkylene,substituted alkylene, cycloalkylene, substituted cylcoalkylene,alkenylene, substituted alkenylene, alkynylene, substituted alkynylene,cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene,heterocyclene, and a covalent bond; Y^(a) and Y^(b) at each separateoccurrence are selected from the group consisting of —C(O)NR′—,—NR′C(O)—, —NR′C(O)NR′—, —C(═NR′)—NR′—, —NR′—C(═NR′)—, —NR′—C(O)—O—,—P(O)(OR′)—O—, —S(O)_(n)CR′R″—, —S(O)_(n)—NR′—, —S—S— and a covalentbond; where n is 0, 1 or 2; and R, R′ and R″ at each separate occurrenceare selected from the group consisting of hydrogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,aryl, heteroaryl and heterocyclic.
 7. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of any of claims 1 to
 6. 8. A method oftreating a bacterial infection in a patient, the method comprisingadministering a therapeutically effective amount of the pharmaceuticalcomposition of claim 7.